Method for selectively separating live cells expressing a specific gene

ABSTRACT

The present invention provides a method for selectively separating live cells which have expressed a specific mRNA comprising: a first step of introducing a marker which label mRNA into cells in a live cell group containing live cells which have expressed a specific mRNA; a second step of labeling said mRNA with said marker to obtain a live cell group containing live cells having the labeled mRNA; and a third step of detecting said labeled mRNA to identify the live cells having the labeled mRNA and separating the identified live cells selectively from said live cell group obtained in said second step.

This is a continuation of U.S. patent application Ser. No. 09/775,818,filed Feb. 5, 2001, now U.S. Pat. No. 6,872,525 the disclosure of whichis incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for selectively separating live cellswhich have expressed a specific gene.

2. Related Background Art

In the case that the translation products of a gene are cell surfacemolecules, a method for selectively separating cells which haveexpressed the specific gene, while being viable, is to make fluorescencelabeled antibodies bind to the surface molecules for labeling the cellsfluorescently, to identify fluorescing cells by flow cytometry, and toseparate the identified cells with a cell sorter (Fluorescence ActivatedCell Sorter, FACS). In addition, the panning method is also knownwherein only the objective cells are absorbed on the bottom surface of adish over which is covered with antibodies specifically binding to thecell surface molecules.

In the case that the translation products of a gene are not cell surfacemolecules but localize in the cells (in the cytoplasm or in organella),the method described above cannot be adopted. In this case, it istheoretically possible to fluorescently label the gene-expressing cellsby introduction of fluorescence-labeled antibodies that are specific tothe molecules localized in the cells into the cells, throughmicroinjection and to separate the objective gene-expressing cells withthe cell sorter described above based on the difference in fluorescenceintensity of the cells with irradiation of laser beam or the like.

However, for the cell labeling method by microinjection described above,the method can not label many cells at once. The number of the cells towhich the labeled antibodies can be introduced for one experiment is atmost ten or less. In addition, it is not easy to introduce the solutionof a polymer with the molecular weight greater than 120,000 like anantibody with high concentration into the cell because of its highviscosity. Therefore, microinjection is impractical to label thesufficient number of objective cells efficiently.

In the case that the translation products of the gene are not cellsurface molecules, but molecules that are liberated into theextracellular fluid and that do not remain in the cell or near the cellmembrane, it is very difficult to selectively trap the molecules toseparate the cells expressing the specific gene, from other moleculeswith the approaches described above. This is because, during the processwhere polypeptide chains generated based on the genetic information arefolded and secreted, their structure changes gradually and from time totime to prevent any known antibodies from binding to the polypeptidechains within or on the surfaces of live cells efficiently. Also, evenin the case that the translation products are present on the surfaces ofcells, it is difficult to selectively separate the cells unless themolecules are specifically present on the surfaces of particular cells.

A typical example, where the situation described above exists and whenit is difficult to separate the objective live cells selectively,includes the case where cells secreting a specific cytokine areselectively separated using the cytokine as a selection marker.

When an antigen invades an organism, helper T cells (CD4+ T cells) thatrecognize the antigen as a foreign matter are activated, and then theywill be differentiated into TH1 and TH2, which have different immunefunctions from each other: TH1 (T Helper 1) which is responsible forcellular immune functions, e.g., activation of macrophages to removeforeign matters by phagocytosis; and TH2 (T Helper 2) which has humoralimmune functions, e.g., activation of B cells to produce antibodymolecules to neutralize foreign matters and (See FIG. 94). TH1 and TH2produce cytokines, interleukin-2 (IL-2) and interleukin-4 (IL-4),respectively. In the healthy state, TH1 and TH2 control each other'sfunctions and keep a balance. However, once this relationship isdisrupted, it causes various infections or autoimmune disorders.

If TH1 or TH2 can be selectively separated and obtained, it will bemedically important, because their application can be contemplated insupplementing immune functions or the like. Thus, a variety of attemptshave been made to find molecules that are present on the surface of TH1or TH2, that can be used for their separation and obtaining.

For example, it has been reported that the tissue infiltration which isdependent on adhesive molecules, P- and E-selectin, is observedspecifically with human TH1 (Austrup, F. et al. Nature, 385, 81–83,1997). This suggests that ligands adhering specifically to the selectinsare present on TH1 cell surfaces. However, when reactivity for P- andE-selectin is examined by flow cytometry, the results are TH1:TH2=131:52for P-selectin, and TH1:TH2=668:88 for E-selectin; therefore, thespecificity is not complete. These results can be interpreted asreflecting the fact that particularly notable P- and E-selectin ligandexpression is induced in TH1 under physiological conditions unique toinflamed tissues (such as skin and joints).

Receptors for CC-chemokine (CCR3), eotaxin, have also been reported tobe present with approximate specificity on human TH2 cell surfaces(Sallusto, F. et al. Science, 277, 1997). However, since CCR3-negative Tcell groups also include IL-4 producing TH2 cells in a proportion of1.9%, the specificity is not complete. Furthermore, the presence of manymore of the same receptors on eosinophil and basophil cell surfaces thanon TH2 raises the risk of possible contamination by cells other than TH2if CCR3+ cells are simply separated from T lymphocytes that have beencrudely purified from blood.

The receptor CCR5 for other CC-chemokines such as MIP-1β and IP10 andthe receptor CXCR3 for the CXC-chemokine SDF-1 have been reported to bepresent with approximate specificity on human TH1 cell surfaces(Loestscher, P. et al., Nature, 391, 344, 1998). However, since one ofthe nine TH2 clones obtained here was CCR5+, the specificity is notcomplete. Furthermore, while TH1 shows higher CXCR3 gene expression andCXC-chemokine dependent migration than TH2, CXCR3 gene expression wasalso confirmed in all of the TH2 clones examined, and therefore, thespecificity is not complete. Moreover, CCR5 and CXCR3 are also presenton neutrophil cell surfaces, and therefore the risk exists of possiblecontamination by neutrophils in CCR5+ or CXCR3+ cells separated from Tlymphocytes that have been crudely purified from blood.

In addition, IL-12 (interleukin-12) receptor (IL-12R) has been reportedto be present with approximate specificity on human TH1 cell surfaces(Rogge, L. et al., J. Exp. Med., 185, 825, 1997). However, while TH1cell surfaces bear high affinity receptors (Kd value=27 pM) and lowaffinity receptors (Kd value=5 nM) for IL-12 at 140 molecules and 450molecules per cell surface, respectively, similar low affinity receptors(Kd value=2 nM) are also present on TH2 cells at 200 molecules per cellsurface. This means that IL-12R cannot be used as a definitive TH1 cellsurface marker. Moreover, since IL-12R is also present on the cellsurfaces of NK cells, the risk exists of possible contamination by NKcells in IL-12R positive cells separated from T lymphocytes that havebeen crudely purified from blood.

IL-18 (interleukin-18) receptor (IL-18R) is another receptor reported tobe present specifically on the cell surface of a TH1 clone establishedfrom transgenic mice with T cell receptors for ovalbumin (Xu, D. et al.,J. Exp. Med., 188, 1485, 1998). However, like IL-18R, the ST2L moleculebelonging to the interleukin-1 receptor (IL-1R) family is also known tobe present on TH2 cell surfaces. Because gene homology within the IL-1Rfamily is particularly high in humans, IL-18R cannot be considered adefinitive cell surface marker in humans and no reports have yet beenpublished on their presence specifically on TH1 cell surfaces. Also,since IL-18R is much more abundantly present on monocyte, neutrophil andNK cell surfaces than on TH1, the risk exists of possible contaminationby cells other than TH1 in IL-18R positive cells separated from Tlymphocytes that have been crudely purified from blood.

The reports cited above suggested that receptors for cytokines,chemokines and the like present on TH1 or TH2 cell surfaces varyconsiderably in terms of amount (number of per cell surface) and quality(affinity of the receptors for their ligands or intracellulartransduction of stimuli upon binding to ligands), and that thedistribution of such receptors therefore highly favors either TH1 orTH2. The reason for the favorableness of cell surface molecules towardeither TH1 or TH2 is believed to arise from the biological environment(physiological conditions) surrounding the helper T cells.

For example IL-12, which is one of the ligands for these cytokinereceptors, is a cytokine secreted by macrophages, etc. upon initialinfection (Walker, W. et al., J. Immunol., 162, 5894, 1999), and IL-18is also known to be produced by activated macrophages and Kupffer cells(Yoshimoto, T. et al., J. Immunol., 161, 3400, 1998). Naturally, both ofthese cytokines have more connections with TH1 than with TH2, in lightof the cellular immunity function of the former, and they are consideredto be factors that perform transduction of physiological informationfrom macrophages to TH1 (i.e., that activate TH1 in the body).

This suggests a connection between activation of macrophages by TH1 andreception of stimuli (IL-12 and IL-18) returned from macrophages,whereby macrophages activated by TH1 eliminate foreign matters in thebody while also activating TH1. As this interdependent relationship isestablished at sites of inflammation in the body, it is fully expectedfor the number of receptors for IL-12 and IL-18 and the activity of thereceptor molecules to increase significantly on TH1 cell surfaces.Further, since TH2 cells are not exposed to the same conditions in thebody as TH1, it is surmised that they have no need to receive IL-12 orIL-18. However, as long as IL-12 or IL-18 receptors are detected evenslightly on TH2 cell surfaces, it cannot be denied that TH2 also has thepotential to respond to IL-12 or IL-18.

It is therefore inconceivable that these cytokine receptors aredefinitive markers that can distinguish TH1 from among TH1 and TH2. Inaddition, since these chemokine and cytokine receptors that arepredominantly distributed on TH1 and TH2 cells are also founddistributed among other cell types such as NK cells, they are consideredimpractical as markers for distinguishing TH1 or TH2 from each other inblood samples. For example, cell specimens containing CD4+ cells (helperT cells) that are separated and purified by common methods from bloodsamples taken from humans usually include contamination by monocytes andgranulocytes, and these false positive cells may be expected to bemistaken for TH1 or TH2.

As stated above, it is, therefore, very difficult to selectivelyseparate TH1 and TH2 cells based on surface molecules. Moreover, sincethe cytokines (IL-2 and IL-4) produced by TH1 and TH2 do not remain inthe cell or near the cell membrane but are liberated into theextracellular fluid, it is difficult to selectively separate TH1 and TH2using these cytokines.

SUMMARY OF THE INVENTION

The present invention has been made in consideration of the above-statedproblems in the prior art. It is an object of the present invention toprovide a separation method which allows one to selectively separate andobtain the objective cells, that is, the cells which have expressed aspecific gene, when there are no cell surface molecules usable asmarkers in the cell, or when the cell surface molecules cannot bedistinguished from each other even if they are present in the cell, oreven when the molecules to be the markers are liberated into theextracellular fluid.

The present inventors have found that the problems in the prior art asdescribed above result from the fact that the translation products of aspecific gene (polypeptide) are used as targets (or markers) to separategene-expressed cells. Based on this finding, the inventors pursuedfurther research, and as a result, have found that if mRNA is used as atarget (marker) which exists mainly on float in the cytoplasm and whichis a transcriptional product of a gene instead of using the translationproduct (polypeptide) as a marker, it is possible to selectivelyseparate the cells which have expressed a specific gene, while beingviable, when there are no cell surface molecules usable as markers inthe cell, or when the cell surface molecules are not ones which arestrictly specific for the objective cells, or even when the molecules tobe the markers are liberated into the extracellular fluid. The presentinvention has thus been accomplished.

Specifically, the present invention provides a method for selectivelyseparating live cells which have expressed a specific gene comprising:

a first step of introducing a marker capable of labeling mRNA into cellsin a live cell group containing live cells which have expressed aspecific mRNA;

a second step of labeling the mRNA with the marker to obtain a live cellgroup containing live cells having the labeled mRNA; and

a third step of detecting the labeled mRNA to identify the live cellshaving the labeled mRNA and separating the identified live cellsselectively from the live cell group obtained in the second step.

In the method for selectively separating live cells which have expresseda specific gene according to the present invention, it is preferablethat the marker in the first step is a probe which has a base sequencecomplementary to the mRNA and has been labeled with a fluorescent dye,the labeled mRNA in the second step is a hybrid of the probe and themRNA, and the selective separation in the third step is performed byirradiating light to the live cell group containing live cells havingthe hybrid, identifying live cells which cause a change in fluorescenceof said fluorescent dye based on formation of the hybrid, and separatingthe identified live cells from the live cell group.

It is also preferable that the probe comprises a first probe and asecond probe, the first probe and the second probe have base sequencescapable of hybridizing to the mRNA adjacently, the first probe islabeled with an energy donor fluorescent dye and the second probe islabeled with an energy acceptor fluorescence dye, and the change influorescence is caused by fluorescence resonance energy transfer (FRET)from the energy donor fluorescence dye of the first probe to the energyacceptor fluorescence dye of the second probe.

In addition, in the method for selectively separating live cells whichhave expressed a specific gene according to the present invention, it ispreferable that the selective separation in the third step of the livecells based on the changes in fluorescence is performed by a cell sorter(FACS).

It is also preferable that the mRNA is an mRNA encoding a cytokine. Itis more preferable that the mRNA is an mRNA encoding interleukin-2(IL-2) and the first probe is a probe having a base sequence set forthin SEQ ID NO: 9 in the Sequence Listing and further that the secondprobe is a probe having a base sequence set forth in SEQ ID NO: 10 inthe Sequence Listing.

It is also preferable that the mRNA is an mRNA encoding interleukin-4(IL-4) and the first probe is a probe having a base sequence set forthin SEQ ID NO: 17 in the Sequence Listing and further that the secondprobe is a probe having a base sequence set forth in SEQ ID NO: 18 inthe Sequence Listing.

In the present invention, it is preferable that the live cellsselectively separated in the third step are T Helper 1 (TH1) or T Helper2 (TH2) cells.

As stated above, according to the present invention it will becomepossible to provide a separation method which allows one to selectivelyseparate and obtain the objective cells, that is, the cells which haveexpressed a specific gene, when there are no cell surface moleculesusable as markers in the cell, or when the cell surface molecules 11, oreven when the molecules to be the markers are liberated into theextracellular fluid.

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying graphs whichare given by way of illustration only, and thus are not to be consideredas limiting the present invention. Further scope of applicability of thepresent invention will become apparent from the detailed descriptiongiven hereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a listing of the entire base sequence of IL-2 mRNA [SEQ ID NO:21] and the base sequences of oligo DNA probes.

FIG. 2 is a listing of the entire base sequence of IL-4 mRNA [SEQ ID NO:22] and the base sequences of oligo DNA probes.

FIG. 3 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe 228–242(D) and the acceptor probe 243–257(A) beingadjacently hybridized to IL-2 RNA.

FIG. 4 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-2 198–212(D) and the acceptor probe IL-2 213–227(A) beingadjacently hybridized to IL-2 RNA.

FIG. 5 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-2 77–91(D) and the acceptor probe IL-2 92–106(A) beingadjacently hybridized to IL-2 RNA.

FIG. 6 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-2 287–301(D) and the acceptor probe IL-2 302–316(A) beadjacently hybridized to IL-2 RNA.

FIG. 7 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-2 342–356(D) and the acceptor probe IL-2 357–371(A) beingadjacently hybridized to IL-2 RNA.

FIG. 8 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-4 70–84(D) and the acceptor probe IL-4 85–99(A) beingadjacently hybridized to IL-4 RNA.

FIG. 9 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-4 119–133(D) and the acceptor probe IL-4 134–148(A) beingadjacently hybridized to IL-4 RNA.

FIG. 10 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-4 176–190(D) and the acceptor probe IL-4 191–205(A) beingadjacently hybridized to IL-4 RNA.

FIG. 11 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-4 265–279(D) and the acceptor probe IL-4 280–294(A) beingadjacently hybridized to IL-4 RNA.

FIG. 12 is a graph of a fluorescence spectrum of a hybrid formed by thedonor probe IL-4 376–390(D) and the acceptor probe IL-4 391–405(A)adjacently hybridized to IL-4 RNA.

FIG. 13 is a chromatogram of HPLC obtained when a mixture solution ofthe donor probe IL-2 342–356(D) and IL-2 RNA was separated by HPLC.

FIG. 14 is a chromatogram of HPLC obtained when a mixture solution ofthe acceptor probe IL-2 357–371(A) and IL-2 RNA was separated by HPLC.

FIG. 15 is a chromatogram of HPLC obtained when a mixture of the donorprobe IL-4 119–133(D) and IL-4 RNA was separated by HPLC.

FIG. 16 is a chromatogram of HPLC obtained when a mixture of theacceptor probe IL-4 134–148(A) and IL-4 RNA was separated by HPLC.

FIG. 17 is a chromatogram of HPLC obtained when a mixture of the donorprobe IL-4 265–279(D) and IL-4 RNA was separated by HPLC.

FIG. 18 is a chromatogram of HPLC obtained when a mixture of theacceptor probe IL-4 280–294(A) and IL-4 RNA was separated by HPLC.

FIG. 19 is a graph showing the amount of IL-2 secreted by Jurkat E6-1cells associated with the expression induction treatment of IL-2,fluorescence micrographs obtained when IL-2 mRNA in the cell extract wasfluorescently detected, and the number of molecules of the intracellularIL-2 mRNA.

FIG. 20 is a set of fluorescence micrographs obtained for those to whichDIG-labeled dUTP and reverse transcriptase were added afteroligonucleotides (oligo dT or oligo dA) had been introduced to IL-2expression-induced cells or IL-2 expression-uninduced cells in the fixedstate, those to which the oligonucleotides were not introducedthereafter, and those to which neither of them was introduced nor wasadded.

FIG. 21 is a set of fluorescence micrographs obtained when hybrids wereformed between IL-2 mRNA in the IL-2 expression-induced cells or IL-2expression-uninduced cells in the fixed state and various probes(non-fluorescent markers), and the hybrids were fluorescently detected.

FIG. 22 is a graph showing the results obtained when hybrids were formedbetween IL-2 mRNA in the IL-2 expression-induced cells or IL-2expression-uninduced cells in the fixed state and various probes(non-fluorescent markers) were fluorescently detected, and thefluorescence intensities were normalized based on the values offluorescence intensity emitted from the fluorescent labeled compoundsformed between total mRNA in said cells and oligo dT.

FIG. 23 is a set of fluorescence micrographs showing D/A, D/D and A/Aimages of hybrids formed by the three components, IL-2 mRNA in IL-2expression-induced cells or IL-2 expression-uninduced cells in the fixedstate, respective donor probes and respective acceptor probes uponexcitation of the donor fluorescent dyes of the hybrids.

FIG. 24 is a graph showing the results obtained when the fluorescence ofthe acceptor fluorescence dyes was measured upon excitation of the donorfluorescent dyes of the hybrids formed by the three molecules, IL-2 mRNAin IL-2 expression-induced cells or IL-2 expression-uninduced cells inthe fixed state, respective donor probes and respective acceptor probes,and the measured fluorescence was standardized based the measured valuesof fluorescence of the acceptor fluorescent dyes upon excitation of theacceptor fluorescent dyes representing all the acceptor probes in saidcells.

FIG. 25 is a set of fluorescence micrographs of D/A, D/D and A/A imagesof a hybrid formed by the three molecules, IL-2 mRNA in IL-2expression-induced cells or IL-2 expression-uninduced cells in theliving state, IL-2 342–356(D) and IL-2 357–371(A), and the correspondingphase contrast micrograph.

FIG. 26 is a dot plot of the results based on forward scattering lightand side scattering light for a cell group where the mixing ratio of theIL-2 expression-induced cells to the IL-2 expression-uninduced cells was100:0 when subjected to flow cytometry (R1 is the region selected forlive cells to be measured).

FIG. 27 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 100:0 when subjected to flowcytometry (R2 is the region selected for fluorescing cells due to FRET).

FIG. 28 is a dot plot of the results based on forward scattering lightand side scattering light for a cell group where the mixing ratio of theIL-2 expression-induced cells to the IL-2 expression-uninduced cells was0:100 when subjected to flow cytometry.

FIG. 29 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 0:100 when subjected to flowcytometry.

FIG. 30 is a dot plot of the results based on forward scattering lightand side scattering light for a cell group where the mixing ratio of theIL-2 expression-induced cells to the IL-2 expression-uninduced cells was50:50 when subjected to flow cytometry (R1 is the region selected forlive cells to be measured).

FIG. 31 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 50:50 when subjected to flowcytometry (R2 is the region selected for fluorescing cells due to FRET).

FIG. 32 is a dot plot of the results based on forward scattering lightand side scattering light for a cell group where the mixing ratio of theIL-2 expression-induced cells to the IL-2 expression-uninduced cells was20:80 when subjected to flow cytometry (R1 is the region selected forlive cells to be measured).

FIG. 33 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 20:80 when subjected to flowcytometry (R2 is the region selected for fluorescing cells due to FRET).

FIG. 34 is a dot plot of the results based on forward scattering lightand side scattering light for the cell group where the mixing ratio ofthe IL-2 expression-induced cells to the IL-2 expression-uninduced cells100:0 when the selective separation was conducted according to flowcytometry by gating with the R1 gate of FIG. 26 and the R2 gate of FIG.27, and the resulting cell group was again subjected to flow cytometry.

FIG. 35 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 100:0 when the selectiveseparation was conducted according to flow cytometry by gating with theR1 gate of FIG. 26 and the R2 gate of FIG. 27, and the resulting cellgroup was again subjected to flow cytometry.

FIG. 36 is a dot plot of the results based on forward scattering lightand side scattering light for the cell group where the mixing ratio ofthe IL-2 expression-induced cells to the IL-2 expression-uninduced cellswas 50:50 when the selective separation was conducted according to flowcytometry by gating with the R1 gate of FIG. 30 and the R2 gate of FIG.31, and the resulting cell group was again subjected to flow cytometry.

FIG. 37 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 50:50 when the selectiveseparation was conducted according to flow cytometry by gating with theR1 gate of FIG. 30 and the R2 gate of FIG. 31, and the resulting cellgroup was again subjected to flow cytometry.

FIG. 38 is a dot plot of the results based on forward scattering lightand side scattering light for the cell group where the mixing ratio ofthe IL-2 expression-induced cells to the IL-2 expression-uninduced cellswas 20:80 when the selective separation was conducted according to flowcytometry by gating with the R1 gate of FIG. 32 and the R2 gate of FIG.33, and the resulting cell group was again subjected to flow cytometry.

FIG. 39 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET for thecell group where the mixing ratio of the IL-2 expression-induced cellsto the IL-2 expression-uninduced cells was 20:80 when the selectiveseparation was conducted according to flow cytometry by gating with theR1 gate of FIG. 32 and the R2 gate of FIG. 33, and the resulting cellgroup was again subjected to flow cytometry.

FIG. 40 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 100:0 before subjectedto flow cytometry.

FIG. 41 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 100:0 when theselective separation was conducted according to flow cytometry by gatingwith the R1 gate of FIG. 26 and the R2 gate of FIG. 27.

FIG. 42 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 50:50 before subjectedto flow cytometry.

FIG. 43 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 50:50 when theselective separation was conducted according to flow cytometry by gatingwith the R1 gate of FIG. 40 and the R2 gate of FIG. 31.

FIG. 44 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 20:80 before subjectedto flow cytometry.

FIG. 45 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 20:80 when theselective separation was conducted according to flow cytometry by gatingwith the R1 gate of FIG. 32 and the R2 gate of FIG. 33.

FIG. 46 is a set of micrographs showing D/A, D/D and A/A images, and thecorresponding phase contrast micrograph of the cell group where themixing ratio of the IL-2 expression-induced cells to theexpression-uninduced cells in the live state was 0:100 before subjectedto flow cytometry.

FIG. 47 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 100:0 before flow cytometrythereof was fixed to a glass-bottomed dish, hybrids were formed betweenthe cellular IL-2 mRNA in the fixed state and RNA probe for IL-2 RNA s,and the hybrids were fluorescently detected.

FIG. 48 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 100:0 was subjected toselective separation according to flow cytometry by gating with the R1gate of FIG. 26 and the R2 gate of FIG. 27, the resulting cell group wasfixed to a glass-bottomed dish, hybrids were formed between the cellularIL-2 mRNA in the fixed state and RNA probe for IL-2 RNA s, and thehybrids were fluorescently detected.

FIG. 49 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 0:100 before flow cytometrythereof was fixed to a glass-bottomed dish, hybrids were formed betweenthe cellular IL-2 mRNA in the fixed state and RNA probe for IL-2 RNA s,and the hybrids were fluorescently detected.

FIG. 50 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 50:50 before flow cytometrythereof was fixed to a glass-bottomed dish, hybrids were formed betweenthe cellular IL-2 mRNA in the fixed state and RNA probe for IL-2 mRNA s,and the hybrids were fluorescently detected.

FIG. 51 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 50:50 was subjected toselective separation according to flow cytometry by gating with the R1gate of FIG. 30 and the R2 gate of FIG. 31, the resulting cell group wasfixed to a glass-bottomed dish, hybrids were formed between the cellularIL-2 mRNA in the fixed state and RNA probe for IL-2 RNA s, and thehybrids were fluorescently detected.

FIG. 52 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 20:80 before flow cytometrythereof was fixed to a glass-bottomed dish, hybrids were formed betweenthe cellular IL-2 mRNA in the fixed state and RNA probe for IL-2 RNA s,and the hybrids were fluorescently detected.

FIG. 53 is a fluorescence micrograph obtained when the cell groupobtained by mixing IL-2 expression-induced cells and IL-2expression-uninduced cells at the ratio of 20:80 was subjected toselective separation according to flow cytometry by gating with the R1gate of FIG. 32 and the R2 gate of FIG. 33, the resulting cell group wasfixed to a glass-bottomed dish, hybrids were then formed between thecellular IL-2 mRNA in the fixed state and RNA probe for IL-2 RNA s, andthe hybrids were fluorescently detected.

FIG. 54 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for lymphocytes separated fromhuman peripheral blood when subjected to flow cytometry.

FIG. 55 is a dot plot of the results of lymphocytes separated from humanperipheral blood that were fluorescently labeled on cell surfacesthereof with a control antibody when subjected to flow cytometry.

FIG. 56 is a dot plot of the results of lymphocytes separated from humanperipheral blood that were fluorescently labeled at CD4 (CD4 FITC) andCD8 (CD8 PE) on cell surfaces thereof when subjected to flow cytometry.

FIG. 57 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for CD4+ cells separated fromhuman peripheral blood with a CD4+ cell separating column when subjectedto flow cytometry.

FIG. 58 is a dot plot of the results of CD4+ cells separated from humanperipheral blood with a CD4+ cell separating column that werefluorescently labeled with a control antibody when subjected to flowcytometry.

FIG. 59 is a dot plot of the results of CD4+ cells separated from humanperipheral blood with a CD4+ cell separating column that werefluorescently labeled at CD4 (CD4 FITC) and CD8 (CD8 PE) on cellsurfaces thereof when subjected to flow cytometry.

FIG. 60 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages of a hybrid formed by the three components, intracellular IL-2mRNA of a CD4+ cell (helper T cell) in the live state, IL-2 342–356(D)and IL-2 357–371(A), and the corresponding phase contrast micrograph.

FIG. 61 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages, and the corresponding phase contrast micrograph, of a cell groupobtained by selective separation of the CD4+ cell group of FIG. 60 inthe live state according to flow cytometry by gating with the R1 gate ofFIG. 62 and the R2 gate of FIG. 63.

FIG. 62 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell group of CD4+ cells(helper T cells) of FIG. 60 when subjected to flow cytometry (R1 is theregion selected for live cells to be measured).

FIG. 63 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, of thecell group of CD4+ cells (helper T cells) of FIG. 60 when subjected toflow cytometry (R2 is the region selected for fluorescing cells due toFRET).

FIG. 64 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell sorter-separated cellgroup of FIG. 61 when subjected to flow cytometry (where R1 is theregion selected for live cells to be measured).

FIG. 65 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell sorter-separated cell group of FIG. 61 when subjected to flowcytometry (R2 is the region selected for fluorescing cells based onFRET).

FIG. 66 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for a cell group of CD4+ cells(helper T cells) with no fluorescent probes introduced, when subjectedto flow cytometry.

FIG. 67 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell group of CD4+ cells (helper T cells) with no fluorescent probesintroduced, when subjected to flow cytometry.

FIG. 68 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages of a hybrid formed by the three components, intracellular IL-4mRNA of CD4+ cells (helper T cells) in the live state, IL-4 265–279(D)and IL-4 280–294(A) and the corresponding phase contrast micrograph.

FIG. 69 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages, and the corresponding phase contrast micrograph, of the cellgroup obtained by selective separation of CD4+ cell group in the livestate of FIG. 68 according to flow cytometry by gating with the R1 gateof FIG. 70 and the R2 gate of FIG. 71.

FIG. 70 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell group of CD4+ cells(helper T cells) in the live state of FIG. 68 when subjected to flowcytometry (R1 is the region selected for live cells to be measured).

FIG. 71 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell group of CD4+ cells (helper T cells) of FIG. 68 when subjected toflow cytometry (R2 is selected for fluorescing cells due to FRET).

FIG. 72 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell sorter-separated cellgroup of FIG. 69 when subjected to flow cytometry (R1 is the regionselected for live cells to be measured).

FIG. 73 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell sorter-separated cell group of FIG. 35 when subjected to flowcytometry (R2 is the region selected for fluorescing cells based onFRET).

FIG. 74 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages of a hybrid formed by the three components, intracellular IL-2mRNA of TH2-induced CD4+ cell in the live state (helper T cell), IL-2342–356(D) and IL-2 357–371(A) and the corresponding phase contrastmicrograph.

FIG. 75 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages, and the corresponding phase contrast micrograph, of the cellgroup obtained by selective separation of CD4+ cell group in the livestate of FIG. 74 according to flow cytometry by gating with the R1 gateof FIG. 76 and the R2 gate of FIG. 77.

FIG. 76 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell group of CD4+ cells(helper T cells) of FIG. 74 when subjected to flow cytometry (R1 is theregion selected for live cells to be measured).

FIG. 77 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell group of CD4+ cells (helper T cells) of FIG. 74 when subjected toflow cytometry (R2 is the region selected for fluorescing cells based onFRET).

FIG. 78 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell sorter-separated cellgroup of FIG. 75 when subjected to flow cytometry (R1 is the regionselected for live cells to be measured).

FIG. 79 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell sorter-separated cell group of FIG. 75 when subjected to flowcytometry (R2 is the region selected for fluorescing cells based onFRET).

FIG. 80 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages of a hybrid formed by the three components, the intracellularIL-4 mRNA of CD4+ cells T1 induction-treated in the live state (helper Tcells), IL-4 265–279(D) and IL-4 280–294(A) and the corresponding phasecontrast micrograph.

FIG. 81 is a set of micrographs showing A/A, D/A and D/D fluorescenceimages, and the corresponding phase contrast micrograph, of a cell groupobtained by selective separation of CD4+ cell group of FIG. 80 in thelive state according to flow cytometry by gating with the R1 gate ofFIG. 82 and the R2 gate of FIG. 83.

FIG. 82 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell group of CD4+ cells(helper T cells) of FIG. 80 when subjected to flow cytometry (R1 is theregion selected for live cells to be measured).

FIG. 83 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity of the energy acceptor fluorescent dye due to FRET, for thecell group of CD4+ cells (helper T cells) of FIG. 80 when subjected toflow cytometry (R2 is the region selected for fluorescing cells based onFRET).

FIG. 84 is a dot plot of the results based on forward scattering light(FSC) and side scattering light (SSC) for the cell sorter-separated cellgroup of FIG. 81 when subjected to flow cytometry (R1 is the regionselected for live cells to be measured).

FIG. 85 is a dot plot of the results based on relative fluorescenceintensity of the energy donor fluorescent dye and relative fluorescenceintensity due of the energy acceptor fluorescent dye due to FRET, forthe cell sorter-separated cell group of FIG. 81 when subjected to flowcytometry (R2 is the region selected for fluorescing cells based onFRET).

FIG. 86 is a set of fluorescence micrographs obtained when the cellgroup of CD4+ cells (helper T cells) of FIG. 60 before flow cytometrythereof was fixed to the bottom of a cover glass chamber, hybrids wereformed between the IL-2 mRNA or IL-4 mRNA of the fixed cells and RNAprobe for IL-2 RNA s or IL-4 RNA probes, respectively, and the hybridswere fluorescently detected.

FIG. 87 is a set of fluorescence micrographs obtained when the cellgroup of cells selectively separated with the cell sorter in FIG. 61 wasfixed to the bottom of a cover glass chamber, hybrids were formedbetween the IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 mRNA of the fixedcells and IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 RNA probe,respectively, and the hybrids were fluorescently detected.

FIG. 88 is a set of fluorescence micrographs obtained when the cellgroup of CD4+ cells (helper T cells) of FIG. 68 before flow cytometrythereof was fixed to the bottom of a cover glass chamber, hybrids wereformed between the IL-2 mRNA or IL-4 mRNA of the fixed cells and RNAprobe for IL-2 RNA s or IL-4 RNA probes, respectively, and the hybridswere fluorescently detected.

FIG. 89 is a set of fluorescence micrographs obtained when the cellgroup of cells selectively separated with the cell sorter in FIG. 69 wasfixed to the bottom of a cover glass chamber, hybrids were formedbetween the IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 mRNA of the fixedcells and IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 RNA probes,respectively, and the hybrids were fluorescently detected.

FIG. 90 is a set of fluorescence micrographs obtained when the cellgroup of CD4+ cells (helper T cells) of FIG. 74 before flow cytometrythereof was fixed to the bottom of a cover glass chamber, hybrids wereformed between the IL-2 mRNA or IL-4 mRNA of the fixed cells and RNAprobe for IL-2 RNA s or IL-4 RNA probes, and the hybrids werefluorescently detected.

FIG. 91 is a set of fluorescence micrographs obtained when the cellgroup of cells selectively separated with the cell sorter in FIG. 75 wasfixed to the bottom of a cover glass chamber, hybrids were formedbetween the IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 mRNA of the fixedcells and IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 RNA probes,respectively, and the hybrids were fluorescently detected.

FIG. 92 is a set of fluorescence micrographs obtained when the cellgroup of CD4+ cells (helper T cells) of FIG. 80 before flow cytometrythereof was fixed to the bottom of a cover glass chamber, hybrids wereformed between the IL-2 mRNA or IL-4 mRNA of the fixed cells and RNAprobe for IL-2 RNA s or IL-4 RNA probes, and the hybrids werefluorescently detected.

FIG. 93 is a set of fluorescence micrographs obtained when the cellgroup of cells selectively separated with the cell sorter in FIG. 81 wasfixed to the bottom of a cover glass chamber, hybrids were formedbetween the IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 mRNA of the fixedcells and IL-2, γ-IF, TNF-β, IL-4, IL-5 or IL-10 RNA probes,respectively, and the hybrids were fluorescently detected.

FIG. 94 is a representation showing the mutual relationship amongdifferent cells that constitute the immune system throughout theirdifferentiation as well as the manner in which the different cellscooperate or restrain each other through cytokines such as interleukinsto maintain homeostatis of immune functions of the living body.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method for selectively separating live cells which have expressed aspecific gene according to the present invention comprises:

a first step of introducing a marker capable of labeling mRNA into cellsin a live cell group containing live cells which have expressed aspecific gene;

a second step of labeling said mRNA with said marker to obtain a livecell group containing live cells having the labeled mRNA; and

a third step of detecting said labeled mRNA to identify the live cellshaving the labeled mRNA and separating the identified live cellsselectively from the said live cell group obtained in the said secondstep.

Markers to be introduced into the cells in the present invention may bethose which can label mRNA and are not particularly limited. The markerproduces labeled mRNA when it binds to mRNA in the cell. When mRNA doesnot exist in the cell, or when the marker is excessively introduced evenwhen mRNA is present, the marker which does not involve a bond with mRNAmay remain in the cell. Then, the markers are preferably detectable onlywhen they have been bound to mRNA, or are those which can be detected todetermine whether they have bound to mRNA or not.

In the present invention, probes which have base sequences complementaryto mRNA and which are labeled with a fluorescent dye (hereinafter called“fluorescence-labeled probe” in some cases) are preferably used asmarkers. These probes form hybrid with the mRNA in the cell. However,there are some probes which do not form hybrids in the cell, and it isnecessary to detect the probes forming hybrids selectively as describedabove. Then, it is preferable to use, as the present probes, those whichcause fluorescence changes based on the formation of hybrids.

For these probes described above, two kinds of probes labeled withdifferent fluorescent dyes from each other are used as a pair. In otherwords, it is preferred to use a probe comprising a first probe and asecond probe: the first probe and the second probe have base sequenceshybridizable with the mRNA adjacently; the first probe is labeled withan energy donor fluorescent dye, and the second probe is labeled with anenergy acceptor fluorescent dye.

When the energy donor fluorescent dye which labels the first probe andthe energy acceptor fluorescent dye which labels the second probe arebrought close to each other at a proper distance (for example, less than8 nm), fluorescence resonance energy transfer (FRET) will be possible.Therefore, two fluorescent dyes in probe should be preferably placed ata distance which allows FRET to occur between the energy donorfluorescent dye and the energy acceptor fluorescent dyes when the threemolecules, the first probe, the second probe and the mRNA form a hybrid.The preferable distance between two types of fluorescent dye in theformed hybrid depends on the kinds of fluorescent dye and sites ofhybridization on mRNA. However, the distance between the two fluorescentdyes is preferably within 20 bases or less, more preferably within 2–4bases. When probes which can generate FRET are designed, for example,Lakowicz, J. R. “Principles of Fluorescence spectroscopy” (1983), Plenumpress, New York may be referred to.

Energy donor fluorescent dyes which may be used in the present inventioninclude4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionicacid and derivatives thereof (for example, Bodipy 493/503 or Bodipy FLavailable from Molecular Probes); tetramethylrhodamine-5-(and-6)isothiocyanate) (TRITC) and derivatives thereof (available fromMolecular Probes).

Energy acceptor fluorescent dyes which may be used in the presentinvention include 1,1′-bis(ε-carboxypentyl)-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate potassium salt and derivativesthereof (for example, Cy3 or Cy5, available from Amersham PharmaciaBioTech). X-rhodamine-5-(and -6)-isothiocyanate (XRITC) and derivativesthereof (available from Molecular Probes);6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid and derivatives thereof (for example, Bodipy 630/650 or Bodipy650/665, available from Molecular Probes).

In the present invention, it is preferable to use Bodipy 493/503 as anenergy donor fluorescent dye, and Cy5 or XRITC as an energy acceptorfluorescent dye.

In the present invention, the number of bases of oligonucleotide to forma probe is not strictly limited. When the number of bases is extremelysmall, e.g., less than 10, it will, however, likely be difficult to forma fully stable hybrid. When the number of bases in a probe is largeexceeding 50, not only the synthesis of the probe is difficult, but alsothe stability of the probe is degraded; and it can take a longer time toform a hybrid.

The number of bases in a probe for hybridization is determined under theconsideration of the conditions of hybridization, such as theconcentration of target mRNA in a live cell to be used and thetemperature in hybridization. Generally, the melting point of a hybridformed with a probe and mRNA is elevated with the increased number ofbases in a probe. For example, if the number of bases in a probe is 15or so, a hybrid is likely to be formed at room temperature withadequately high efficiency, but at 37° C., the hybrid is not to beformed with high efficiency. In order to detect a hybrid at 37° C., itis desirable to use probes with the length of 15 bases or more,preferably 20 bases or more.

On the other hand, the ratio of hybrid formation decreases as the numberof bases increases when the number of bases of a probe is in the rangeof 15–20. For example, at room temperature, the time necessary tocomplete the hybridization between a probe of 20 bases and mRNA isseveral times longer than the time required for a probe of 15 bases.

Taken these requirements together, it is further preferable that thenumber of bases in a probe is 10–50, more preferably 15–20.

When the probes are designed, it is also important what site in the mRNAprobes hybridize to, as well as the number of bases in a as describedabove. That is, mRNA itself is a molecule with complicated secondary andtertiary structure. Thus, even if the probe to be used has a basesequence complementary to a particular site of the mRNA, an obstructionfor the probe to hybridize to the site often occurs in the secondary andtertiary structure when the site interacts with other sites of the mRNA.In the present invention, therefore, the sites where the probeshybridize to have to be selected.

The sites for hybridization are determined, for example, by approachesdescribed below. First, base sequences of the objective mRNA areobtained from a database. If databases are not available, the basesequences of the mRNA may be determined by well-known methods. Accordingto the information, a secondary structure of the mRNA is simulated. Forthis simulation, it is possible to use commercially available computerprograms for predicting the secondary structure of RNA, such as DNAsis(Hitachi Software Engineering Inc.). Using the obtained secondarystructure, a site with appropriate number of bases, which include thebase sequences in the site free from obstruction for hybridization, isselected; an oligonucleotide having a base sequence complementary to theselected base sequence is synthesized; the synthesized oligonucleotideis fluorescently labeled; and the oligonucleotide is used as a probe.

After several objective sites having 30–40 bases for hybridization areselected, it is preferable that each site is subdivided into two parts(each 15–20 bases). Oligonucleotides having base sequences respectivelycomplementary to the subdivided parts are synthesized, and thenfluorescently labeled to prepare the first probe and the second probefor use.

In order to select a desirable set of probes from several sets of probesselected and synthesized as above, the following method can be used: thefirst probe and the second probe are mixed and the fluorescence spectrais measured; then, the objective mRNA is added to the mixed solution toobserve any changes in the fluorescence spectra. When the first probe,the second probe and mRNA is formed into a hybrid, FRET occurs betweentwo types of fluorescent dyes; as a result, the fluorescence intensityof the energy donor dye decreases, while a fluorescence spectrum isobtained where the fluorescence intensity of the energy acceptor dye isincreased. The aforementioned operation is carried out on several setsof first and second probes; the changes in fluorescence spectra arecompared; and a set of probes with a greater change is selected. Theobjective mRNA used for these methods can be synthesized by in vitrotranscription reaction using a recombinant plasmid DNA which includescDNA corresponding to the mRNA.

Then, hybrids and free probes are separated from each other using highperformance liquid chromatography (HPLC) or the like to correctlyevaluate the efficiency of each probe to hybridize to the objectivemRNA, each probe and each objective RNA are mixed and reacted in anaqueous solution.

The method to design the desirable fluorescence-labeled probes adoptedin the present invention is described above in detail. Suchfluorescence-labeled probe is one of the preferable forms of markers tobe used in the present invention. In the present invention, aftermarkers have been prepared, they may be introduced into the live cellswhich have expressed a specific mRNA. As there are no limitations to themethods for introducing markers into the live cells, well-known methodsare available, including microinjection, electroporation, andlipofection methods. In the present invention, the electroporationmethod is preferable, because the method can introduce markers into morethan 10,000,000 of live cells in a short time at once.

After the markers have been introduced into the live cells, mRNA islabeled with the markers in the cells. Fluorescence-labeled probes areused as markers, which specifically hybridize to the corresponding mRNA.The conditions for hybridization are not limited specifically, but, forexample, live cells which have been introduced fluorescent labeledprobes, may be retained at room temperature at least for a few minutes.

After the cellular mRNA is labeled, live cells containing the labeledmRNA are identified by detecting the labeled mRNA, and the identifiedcells are selectively separated. In the present invention, there are nolimitations to the method to detect the labeled mRNA. All of the livecell groups of which markers have been introduced do not always expressthe objective mRNA. In addition, when the marker has been introducedexcessively in amount into the cells expressing the mRNA, the markerwhich does not bind to the mRNA will remain in the cell. Therefore, itis necessary to detect the labeled mRNA, i.e., markers bound to themRNA, specifically among the unbound and free markers.

As a method for easily and highly sensitively detecting labeled mRNA inthe presence of markers unbound to the mRNA, the first probe labeledwith an energy donor dye and the second probe labeled with an energyacceptor dyes are preferably used together. These probes are introducedinto the live cells to keep the live cells under the conditions wherethe probes and the mRNA can hybridize, the excitation light of theenergy donor fluorescent dye of the first probe is irradiated to thelive cell group, whereby the fluorescence from the energy acceptorfluorescent dye of the second probe is observed based on FRET, andfinally the fluorescent labeled mRNA is detected specifically.

Although irradiation of excitation light excites the energy donor dye ofthe first probe whether the probe hybridizes to the objective mRNA ornot, only when the both of the first and second probes hybridizeadjacently to the same mRNA, FRET occurs, resulting in emission offluorescence from the energy acceptor fluorescent dye. That is, theFRET-fluorescence from the acceptor fluorescent dye indicates that thefirst probe and the second probe are adjacent on the objective mRNA,showing that the objective genes are expressed in the cells.

The live cells which have expressed specific genes detected in this wayare selectively separated, but there are no limitations for thisseparating method. In the present invention, when fluorescence-labeledprobes are used, it is preferable to use a cell sorter (FluorescenceActivated Cell Sorter, FACS) to detect live cells, which expressspecific genes, and then to selectively separate them.

In general, an apparatus consisting of a flow cytometer and a celldispenser is called a cell sorter. Individual cells, which have beenstained with fluorescence-labeled probes, are exposed to laser beam onthe way of a flow path, resulting in the light scattering andfluorescence from the cell. The intensity of light scattering (forwardscattering light or side scattering light) and fluorescence are measuredfor each cell and the results for a number of cells are displayed, forexample, as a frequency distribution diagram (dot plot). Then, the cellsemitting fluorescence with the desired extent are collected by gating.The method as mentioned above is called flow cytometry.

In the present invention, when live cells which have expressed aspecific gene are selectively separated with the cell sorter usingfluorescent labeled probes which can cause FRET, the following methodcan be applied. In the cell sorter, laser exciting energy donor isirradiated to each cell, and fluorescence intensities of energy donorfluorescent dyes (for example, Bodipy 493/503) as well as on relativefluorescence intensities of energy acceptor dyes (for example, Cy5) areobtained. Each cell is represented as a dot according to the intensityof the donor and acceptor fluorescence, in a diagram where thehorizontal and vertical axis represents intensity of donor and acceptorfluorescence, respectively. Then, in the dot-plotted diagram, the cellsemitting relatively high fluorescence intensity on the vertical axis areselected and these cells are designated as a region in the diagram, R2.Next, obtaining the intensity of forward scattering light (FSC), andside scattering light (SSC) representing the size of live cells to bemeasured, and the complexities of cellular intrastructures,respectively, each cell is represented as a dot in a diagram accordingto the value of FSC and SSC, on the horizontal and vertical axis,respectively. In the dot-plotted diagram, a region representing thecells emitting FSC as well as SSC with the desirable extent, is selectedand designated as R1. After a cell sorter is set so that only live cellssuitable for both conditions of R1 and R2 are collected, the cellsexpressing specific mRNA are selectively separated.

By using the methods as described, various types of live cells as wellas various type of mRNA are objectives of selective separation. TH1 andTH2 derived from helper T cells which have been activated by recognizingantigens as foreign do not have any crucial cell surface antigens(markers) to be distinguished from each other. Further, cytokinesproduced by TH1 and TH2, IL-2 and IL-4, respectively, do not remain inthe cell or near the cell membrane and are liberated to theextracellular fluid. Thus, TH1 or TH2 is an ideal objective to which theselective separation of the present invention is applied. That is, theselective separation method according to the present invention ispreferably used for live cell groups containing live cells having mRNAencoding cytokines.

Especially, it is preferable that live cell groups containing live cellshaving mRNA encoding interleukin-2 (IL-2) are applied to this method ofthe present invention. To separate live cells expressing IL-2 mRNA, thefirst probe labeled with the energy donor fluorescent dye having a basesequence set forth in SEQ ID NO: 9 in the Sequence Listing and thesecond probe labeled with the energy acceptor fluorescent dye having abase sequence set forth in SEQ ID NO: 10 in the Sequence Listing areused, and FRET generated by these probes is utilized as an index forselective separation.

The base sequence set forth in SEQ ID NO: 9 in the Sequence Listing iscomplementary to the base sequence, 342–356 in the base sequence of mRNAencoding IL-2, while the base sequences set forth in SEQ ID NO: 10 inthe Sequence Listing is complementary to the base sequence, 357–371 inthe base sequence of mRNA encoding IL-2. When the first probe labeledwith the energy donor fluorescent dye are made adjacent to the secondprobe labeled with the energy acceptor fluorescent dye at the positionsindicated above of the same mRNA molecule, the detection of theFRET-fluorescence can be carried out with high sensitivity.

Furthermore, it is preferable that live cell groups containing livecells having mRNA encoding interleukin-4 (IL-4) are are applied to thismethod of the present invention. To separate live cells expressing IL-4mRNA, the first probe labeled with the energy donor fluorescent dyehaving a base sequence set forth in SEQ ID NO: 17 in the SequenceListing and the second probe labeled with the energy acceptorfluorescent dye having a base sequence set forth in SEQ ID NO: 18 in theSequence Listing are used, and FRET generated by these probes isutilized as an index for separation.

The base sequence set forth in SEQ ID NO: 17 in the Sequence Listing iscomplementary to the base sequence, 265–279 in the base sequence of mRNAencoding IL-4, while the base sequences set forth in SEQ ID NO: 18 inthe Sequence Listing is complementary to the base sequence, 280–294 inthe base sequence of mRNA encoding IL-4. When the first probe labeledwith the energy donor fluorescent dye are made adjacent to the secondprobe labeled with the energy acceptor fluorescent dye at the positionsindicated above of the same mRNA molecule, the detection of theFRET-fluorescence can be carried out with high sensitivity.

EXAMPLES

The present invention will be explained in greater detail by way ofpreferred examples hereinbelow. However, the present invention shouldnot be limited to these examples.

(1) Preparation of Fluorescent Labeled Markers of OligonucleotidesComplementary to IL-2 mRNA and IL-4 mRNAP

Five sites, 30 bases each, were selected on the base sequences of IL-2or IL-4 mRNA. Each 30 bases-site was divided into two halves (15bases-site). An oligonucleotide complementary to each 15 bases-site wasdesigned as a DNA probe for each site and labeled with a fluorescentdye, Bodipy493/503, Cy5, or XRITC.

The DNA probe was synthesized using a DNA/RNA synthesizer (Perkin Elmer:Model 394 or Perceptive: Model 18909), by β-cyanoethylamidethod. Theentire base sequence of IL-2 mRNA and base sequences of the oligo DNAprobes are shown in FIG. 1, and the entire base sequence of IL-4 mRNAand base sequences of the oligo DNA probes are shown in FIG. 2. The basesequences of the designed 10 types of oligo DNA probes for IL-2 mRNA(SEQ ID NOs: 1–10) and the base numbers (hybridized positions) on IL-2mRNA to which the oligo DNA probes hybridize are shown in Table 1. Thebase sequences of 10 types of oligo DNA probes for IL-4 mRNA (SEQ IDNOs: 11–20) and the base numbers (hybridized positions) on IL-4 mRNA towhich the oligo DNA probes hybridize are shown in Table 2.

TABLE 1 Hybridized SEQ ID NO: Base Sequence Position* SEQ ID NO: 15′-GTAAAACTTAAATGT-3′ 228–242 SEQ ID NO: 2 5′-GGCCTTCTTGGGCAT-3′ 243–257SEQ ID NO: 3 5′-TTTGGGATTCTTGTA-3′ 198–212 SEQ ID NO: 45′-GAGCATCCTGGTGAG-3′ 213–227 SEQ ID NO: 5 5′-GCAAGACTTAGTGCA-3′ 77–91SEQ ID NO: 6 5′-CTGTTTGTGACAAGT-3′  92–106 SEQ ID NO: 75′-GGTTTGAGTTCTTCT-3′ 287–301 SEQ ID NO: 8 5′-AGCACTTCCTCCAGA-3′ 302–316SEQ ID NO: 9 5′-CCTGGGTCTTAAGTG-3′ 342–356 SEQ ID NO: 105′-ATTGCTGATTAAGTC-3′ 357–371 *Base number of IL-2 mRNA to which eachprobe hybridizes.

TABLE 2 Hybridized SEQ ID NO: Base Sequence Position* SEQ ID NO: 115′-CAGTTGGGAGGTGAG-3′ 70–84 SEQ ID NO: 12 5′-GAACAGAGGGGGAAG-3′ 85–99SEQ ID NO: 13 5′-CGTGGACAAAGTTGC-3′ 119–133 SEQ ID NO: 145′-TATCGCACTTGTGTC-3′ 134–148 SEQ ID NO: 15 5′-CTGTGAGGCTGTTCA-3′176–190 SEQ ID NO: 16 5′-ACAGAGTCTTCTGCT-3′ 191–205 SEQ ID NO: 175′-AGCCCTGCAGAAGGT-3′ 265–279 SEQ ID NO: 18 5′-CCGGAGCACAGTCGC-3′280–294 SEQ ID NO: 19 5′-CCGTTTCAGGAATCG-3′ 376–390 SEQ ID NO: 205′-GAGGTTCCTGTCGAG-3′ 391–405 *Base number of IL-4 mRNA to which eachprobe hybridizes.

In (4A) described below, oligo DNAs corresponding SEQ ID NOs: 1–10 werelabeled with Bodipy493/503 at the 5′ end, and were used as probes. In(8), unlabeled oligo DNAs corresponding SEQ ID NOs: 1–10 were used asprobes. In (9), oligo DNAs corresponding SEQ ID NOs: 1, 3, 6, 7, or 9were labeled with Bodipy493/503 at the 5′ end, and oligo DNAscorresponding SEQ ID NOs: 2, 4, 6, 8, or 10 were labeled with XRITC atthe linkage between the 4th nucleotide and the 5th from the 3′ end andwere used as probes. In (11), oligo DNAs corresponding SEQ ID NOs: 1, 3,5, 7, or 9 were labeled with Bodipy493/503 at the 5′ end, and oligo DNAscorresponding SEQ ID NOs: 2, 4, 6, 8, or 10 were labeled with Cy5 at thelinkage between the 4th nucleotide and the 5th from the 3′ end and wereused as probes. The labeling of oligo DNAs with Bodipy493/503, XRITC, orCy5 were performed as described in (a)–(c).

In the present invention, the oligo DNA probes, which are labeled withenergy donor fluorescent dyes, are sometimes abbreviated as donorprobes, while oligo DNA probes, which are labeled with energy acceptorfluorescent dyes are sometimes abbreviated as acceptor probes. Theprobe, corresponding SEQ ID NO: 1 was labeled with an energy donorfluorescent dye, is complementary to the sequence, 228–242 of IL-2 mRNA.Thus, the probe may sometimes be referred to as IL-2 228–242(D) (D meansa donor). The probe, corresponding SEQ ID NO: 2 was labeled with anenergy acceptor fluorescent dye, is complementary to the sequence,243–257 of IL-2 mRNA. Thus, the probe may be sometimes referred to asIL-2 243–257(A) (A means an acceptor). In addition, when the probe isnot labeled with a fluorescent dye, it is simply referred to as IL-2228–242. Thus, the probes used in (9) and (11) may be represented by thenames of probes shown in Table 2 hereunder.

TABLE 3 SEQ ID NO: Base sequence Name of Probes SEQ ID NO: 15′-GTAAAACTTAAATGT-3′ IL-2 228–242 (D) SEQ ID NO: 25′-GGCCTTCTTGGGCAT-3′ IL-2 248–257 (A) SEQ ID NO: 35′-TTTGGGATTCTTGTA-3′ IL-2 198–212 (D) SEQ ID NO: 45′-GAGCATCCTGGTGAG-3′ IL-2 213–227 (A) SEQ ID NO: 55′-GCAAGACTTAGTGCA-3′ IL-2 77–91 (D) SEQ ID NO: 6 5′-CTGTTTGTGACAAGT-3′IL-2 92–106 (A) SEQ ID NO: 7 5′-GGTTTGAGTTCTTCT-3′ IL-2 287–301 (D) SEQID NO: 8 5′-AGCACTTCCTCCAGA-3′ IL-2 302–316 (A) SEQ ID NO: 95′-CCTGGGTCTTAAGTG-3′ IL-2 342–356 (D) SEQ ID NO: 105′-ATTGCTGATTAAGTC-3′ IL-2 367–371 (A)

IL-4 probes are sometimes represented in the same manner as above. Forexample, the probes used in (3B) as described below may be representedby the names of probes shown in Table 4 hereunder.

TABLE 4 SEQ ID NO: Base Sequence Name of Probes SEQ ID NO: 115′-CAGTTGGGAGGTGAG-3′ IL-4 70–84 (D) SEQ ID NO: 12 5′-GAACAGAGGGGGAAG-3′IL-4 85–99 (A) SEQ ID NO: 13 5′-CGTGGACAAAGTTGC-3′ IL-4 119–133 (D) SEQID NO: 14 5′-TATCGCACTTGTGTC-3′ IL-4 134–148 (A) SEQ ID NO: 155′-CTGTGAGGCTGTTCA-3′ IL-4 176–190 (D) SEQ ID NO: 165′-ACAGAGTCTTCTGCT-3′ IL-4 191–205 (A) SEQ ID NO: 175′-AGCCCTGCAGAAGGT-3′ IL-4 265–279 (D) SEQ ID NO: 185′-CCGGAGCACAGTCGC-3′ IL-4 280–294 (A) SEQ ID NO: 195′-CCGTTTCAGGAATCG-3′ IL-4 376–390 (D) SEQ ID NO: 205′-GAGGTTCCTGTCGAG-3′ IL-4 391–405 (A)(a) Preparation of Donor Probes (Bodipy 493/503-Labled)

2.5 mg of NHSS(N-Hydroxysulfosuccinimide sodium salt) in 30 μl ofsterilized water, 5 mg of EDAC[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] in 50 μl of sterilizedwater, and 1 mg of Bodipy493/503 propionic acid dissolved in 50 μl ofDMF were mixed and reacted with at room temperature for 30 minutes.

An oligo DNA with the base sequence described above (lyophilizedproduct), which a hexylamino group was introduced to the 5′ end using6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-(N,N-di-isopropyl)-phosphoroamidite,a 5′ end aminating agent, was dissolved in 200 μl of 0.5 M ofNa₂HCO₃/NaH₂CO₃ buffer solution (pH 9.3). These were mixed and reactedovernight in the dark.

After the reacted solution was gel filtrated to remove unreacted dyes,the reaction solution was subjected to reversed phase high performanceliquid chromatography (HPLC) with CAPCELL PACK18 (Shiseido Inc., Columnsize: 6 mm in inner diameter×250 mm in length), and the fractions withabsorption at 260 nm and 493 nm were recovered and lyophilized. HPLC wasperformed under the following condition, flow rate; 1 ml/minute,temperature; 40° C., the mobile phase was the mixture of solution A (5%CH₃CN containing 5 mM of TEAA) and solution B (40% CH₃CN) and theconcentration gradient of CH₃CN was generated by increasing theconcentration of solution B from 30% to 80 in 20 minutes.

(b) Preparation of Acceptor Probes (Cy5-Labeled)

Cy5 dye in one tube (Amersham, Fluorolink Cat.No.PA25001) was dissolvedin 100 μl of sterilized water. An oligo DNA with the base sequencedescribed above (lyophilized product) which a hexylamino group wasintroduced into the linkage between the 4th nucleotide and the 5th fromthe 3′ end using a Uni-Link AminoModifier (Clontech Inc.), was dissolvedin 200 μl of Na₂HCO₃/NaH₂CO₃ buffer solution (0.5 M, pH 9.3). These weremixed in the dark, and reacted overnight.

After the reacted solution was gel filtrated to remove unreacted dyes,the reaction solution was subjected to reversed phase high performanceliquid chromatography (HPLC) with CAPCELL PACK18 (Shiseido Inc., Columnsize: 6 mm in inner diameter×250 mm in length), and the fractions withabsorption at 260 nm were recovered. HPLC was performed under thefollowing condition, flow rate; 1 ml/minute, temperature; 40° C., themobile phase was the mixture of solution A (5% CH₃CN containing 5 mM ofTEAA) and solution B (40% CH₃CN) and the concentration gradient of CH₃CNwas generated by increasing the concentration of solution B from 15% to60 in 20 minutes. When the absorption spectra of the recovered fractionswas measured in the range of 220–700 nm, the maximum absorption wasobserved between 650–700 nm, indicating a typical property of Cy5. Andthen the fractions were lyophilized.

(c) Preparation of Acceptor Probes (XRITC-Labeld)

One hundred microliters of XRITC dye solution (Solvent: 100% DMSO,Perkin Elmer, ROX-NHS) was reacted with an oligo DNA having the basesequence described above where a hexylamino group was introduced asdescribed in (B). The reaction product was applied to reversed phasehigh performance liquid chromatography; a fraction with an absorptionband at 260 nm was recovered; the absorption spectrum was measured inthe range between 220–650 nm; after the maximum absorption of XRITC wasobserved in the range between 550–600 nm, the recovered fraction waslyophilized.

(2A) In Vitro Synthesis of Human IL-2 RNA

In order to obtain human IL-2 RNA having a base sequence equivalent tohuman IL-2 mRNA, an IL-2 cDNA fragment was cleaved out with restrictionenzyme pst I from pTCGF-II (ATCC#39673), a plasmid containing a humanIL-2 cDNA and was linked to the pst I site of pBluescript KS(+) aplasmid vector for RNA synthesis using Ligation kit version 2 (Takara)so that the cDNA would be located in the downstream of T3 promoter. Theobtained recombinant plasmid was introduced into competent cells of E.coli JM109 strain (Takara Co.), and the transformants of the E. coliobtained were cultured, 46.2 μg of the plasmid DNA was extracted andpurified from 100 ml of the bacterial culture using a Plasmid Midi Kit(QIAGEN).

The recombinant plasmid was linearized by restriction enzyme Sma Idigestion to prepare the template for the synthesis of human IL-2 RNA.The enzyme proteins in the plasmid solution was degraded with proteinaseK and denatured/removed with phenol/chloroform. The RNA synthesis wasperformed using 0.66 μg of the purified template with the basecomposition, A (adenine):C (cytosine):G (guanine):U (uracil),35:18:14:32%. A, C, G and U were added to the template at the finalconcentrations of 105, 54, 42 and 96 mM, respectively together with T3RNA polymerase according to the aid of an in vitro transcription kit(Megascript T3 Kits, Ambion). Polymerase reaction was carried out at 37°C. for 6 hours to synthesize human IL-2 RNA. After the reaction wasover, the RNA was purified as follows. The template DNA was decomposedwith DNase I (Megascript T3 Kits, Ambion Inc.), the enzyme proteins inthe transcription reaction solution was denatured/removed withphenol/chloroform. To the obtained RNA solution was added an equalvolume of isopropanol, and human IL-2 RNA was recovered as a precipitateby centrifugation (14 krpm, for 7 minutes), while the respectivenucleotides which were unreacted enzyme substrates were removed. Thehuman IL-2 RNA precipitate (139 μg) rinsed once with 70% ethanol wasdissolved in RNase-free water (Megascript T3 Kits, Ambion) so that 5μg/μl of human IL-2 RNA solution was prepared to use for the subsequenthybridization experiments.

(2B) In Vitro Synthesis of Human IL-4 RNA

Next, in order to obtain human IL-4 RNA a having base sequenceequivalent to human IL-4 mRNA, human IL-4 cDNA fragment was cleaved outwith restriction enzymes BamHI and Xho I from pcD-hIL-4 (ATCC#57593), aplasmid DNA containing human IL-4 cDNA. The cDNA fragment was linked tothe BamHI and Xho I sites of a pBluescript KS(+), a plasmid vector forRNA synthesis using Ligation kit version 2 (Takara) so that the cDNAwould be located in the downstream of T3 promoter. The obtainedrecombinant plasmid was introduced into competent cells of E. coli JM109strain (Takara Co.); the transformants of the E. coli obtained werecultured; and 152 μg of plasmid DNA was extracted and purified from 100ml of the bacterial culture using a Plasmid Midi Kit (QIAGEN).

The recombinant plasmid was linearized by digestion with restrictionenzyme Sma I to prepare the template for the synthesis of human IL-4RNA. The enzyme proteins in the plasmid solution was degraded withproteinase K and denatured/removed with phenol/chloroform. The RNAsynthesis was performed using 0.46 μg of the purified template with thebase composition, A:C:G:U, 29:24:21:26%. A, C, G and U were added to thetemplate at the final concentrations of 87, 72, 63 and 78 mM,respectively together with T7 RNA polymerase according to the aid of anin vitro transcription kit (Megascript T7 Kits, Ambion). Polymerasereaction was carried out at 37° C. for 6 hours to synthesize human IL-4RNA. After the reaction was over, the RNA was purified as follows. Thetemplate DNA was decomposed with DNase I (Megascript T7 Kits, AmbionInc.), the enzyme proteins in the transcription reaction solution wasdenatured/removed with phenol/chloroform. To the obtained RNA solutionwas added an equal volume of isopropanol, and human IL-4 RNA wasrecovered as a precipitate by centrifugation (14 krpm, for 7 minutes),while the free nucleotides, unreacted enzyme substrates, were removed.The human IL-4 RNA precipitate (139 μg) rinsed once with 70% ethanol wasdissolved in RNase-free water (Megascript T7 Kits, Ambion) so that 5μg/μl of human IL-4 RNA solution was prepared to use for the subsequenthybridization experiments.

(3A) Changes in Fluorescence Spectra by Hybridization of FluorescentLabeled Probes to Human IL-2 RNA

In order to measure changes in fluorescence spectra due to fluorescenceresonance energy transfer (FRET) caused by hybridization of donor probesand acceptor probes to adjacent sites on IL-2 RNA, a pair of 300 nM(final concentration) of Bodipy 493/503-labeld donor probes andXRITC-labeled acceptor probes, and human IL-2 RNA were mixed in 100 μlof 1×SSC solution (150 mM sodium chloride, 17 mM citric acid, pH 7.0),and allowed to stand at room temperature for 15 minutes, and thenfluorescence spectra were measured. As combinations of donor probes andacceptor probes, IL-2 228–242(D) and IL-2 243–257(A), IL-2 198–212(D)and IL-2 213–227(A), IL-2 77–91(D) and IL-2 92–106(A), IL-2 287–301(D)and IL-2 302–316(A), and IL-2 342–356(D) and IL-2 357–371(A), were used.As a control, the fluorescence spectrum of 300 nM of the probe alone wasalso measured. The conditions for fluorescence spectrum measurement wereas follows:

-   Fluorospectrophotometer: F4500 (Hitachi)-   108 Excitation wavelength: 480 nm-   Fluorescence-measurement Wavelength: 500–750 nm-   Temperature: room temperature

For all the combinations examined, changes in fluorescence spectra wereobserved, i.e., the intensity of donor fluorescence decreased whileacceptor fluorescence (580–650 nm) increased with donor excitation dueto the addition of human IL-2 RNA to the probe solution. See FIGS. 3–7.FIGS. 3, 4, 5, and 6 show fluorescence spectra when the combinations ofIL-2 228–242 (D) and IL-2 243–257(A), IL-2 198–212(D) and IL-2213–227(A), IL-2 77–91(D) and IL-2 92–106(A), IL-2 287–301(D) and IL-2302–316(A), and IL-2 342–356(D) and IL-2 357–371(A) were used. As seenfrom the comparison among FIGS. 2–6, the extent of changes influorescence spectra was different from each other among thecombinations of probes, the most remarkable change occurred when thecombination of IL-2 342–356(D) and IL-2 357–371(A) was used.

(3B) Changes in Fluorescence Spectra by Hybridization of FluorescentLabeled Probes to Human IL-4 RNA

In order to measure changes in fluorescence spectra due to fluorescenceresonance energy transfer (FRET) caused by hybridization of donor probesand acceptor probes to adjacent sites on IL-4 RNA, a pair of 300 nM(final concentration) of Bodipy 493/503-labed donor probes andCy5-labeled acceptor probe, and human IL-4 RNA were mixed in 100 μl of1×SSC solution (150 mM sodium chloride, 17 mM citric acid, pH 7.0), andallowed to stand at room temperature for 15 minutes, and thenfluorescence spectra were measured. As combinations of donor probes andacceptor probes, IL-4 70–84(D) and IL-4 85–99(A), IL-4 119–133(D) andIL-4 134–148(A), IL-4 176–190(D) and IL-4 191–205(A), IL-4 265–279(D)and IL-4 280–294(A), and IL-4 376–390(D) and IL-4 391–405(A), were used.As a control, the fluorescence spectrum of 300 nM of the probe describedabove alone was also measured. The conditions for measurement were asfollows:

-   Fluorospectrophotometer: F4500 (Hitachi)-   Excitation wavelength: 480 nm-   Fluorescence-measurement Wavelength: 500–750 nm-   Temperature: room temperature

For all the combinations examined, changes in fluorescence spectra wereobserved, i.e., the intensity of donor fluorescence decreased whileacceptor fluorescence (650–700 nm) increased with donor excitation dueto the addition of human IL-4 RNA to the probe solution. See FIGS. 8–12.FIGS. 8, 9, 10, 11 and 12 show fluorescence spectra when thecombinations of IL-4 70–84 (D) and IL-4 85–99(A), IL-4 119–133(D) andIL-4 134–148(A), IL-4 176–190(D) and IL-4 191–205(A), IL-4 265–279(D)and IL-4 280–294(A), and IL-4 376–390(D) and IL-4 391–405(A) were used.As seen from the comparison among FIGS. 8–12, the extent of changes influorescence spectra was different from each other among thecombinations of probes, the most remarkable change occurred when thecombination of IL-4 265–279(D) and IL-4 280–294(A) was used. The resultsof the measurements are shown as the relative fluorescence intensity ofCy5 to Bodipy493–503 by excitation of Bodipy493–503 (Cy5 fluorescenceintensity/Bodipy493–503 fluorescence intensity) in Table 5.

TABLE 5 Ratio of Fluorescence Probe Combination Intensities IL-4 70–84(D) and IL-4 85–99  1.3% (A) IL-4 119–133 (D) and IL-4  2.5% 134–148 (A)IL-4 176–190 (D) and IL-4 22.4% 191–205 (A) IL-4 265–279 (D) and IL-414.5% 280–294 (A) IL-4 376–390 (D) and IL-4  1.5% 391–405 (A)(4A) Measurement of Hybridization Efficiency of Probes to Human IL-2 RNAby HPLC

Each donor probe (3 pmol) wherein an oligo DNA corresponding SEQ ID NO:1–10 was labeled with Bodipy493/503 was mixed with an equimolar amountof human IL-2 RNA synthesized in (2A) in 10 μl of 1×SSC solution (150 mMsodium chloride, 17 mM sodium citrate, pH 7.0), and the mixture wasallowed to stand at room temperature for 15 minutes. Subsequently,hybrids consisting of human IL-2 RNA and probes were separated from freeprobes by high performance liquid chromatography (HPLC) usingdifferences in retention time under the following conditions, i.e.,retention time is about 4–5 minutes and 7.5 minutes for free probes andhybrids, respectively.

-   Column: TSKgel DRAE-NPR (Toso Inc., 4.6 mm in inner Diameter×35 mm    in total length)-   Flow Rate: 1 ml/minute-   Temperature: 25° C.-   Mobile phase: Solution A: 20 mM Tris-HCl (pH 9.0),-   Solution B: 0.5 mM NaCl, 20 mM Tris-HCl (pH 9.0)

HPLC was performed in the concentration gradient manner. The mobilephase was the mixture of solution A and solution B and the concentrationgradient of NaCl was generated by increasing the concentration ofsolution B from 25% to 100% so that the concentration of NaCl changedfrom 0.125 M to 0.5M in 10 minutes. Absorbance at 260 nm for nucleicacids and fluorescence intensity at 515 nm with the excitation at 475 nmfor Bodipy493/503 were monitored simultaneously on eluted fractions. Thefractions with the absorbance at 260 nm as well as the fluorescence at515 nm were regarded as the ones of hybrids. The relative fluorescenceintensity of the hybrid fractions to all the fractions in thefluorescence chromatogram was estimated and used as an index for theefficiency of hybridization

FIG. 13 shows a HPLC chromatogram when IL-2 342–356 (D) was mixed withIL-2 RNA. FIG. 14 shows a HPLC chromatogram when IL-2 357–371(D) wasmixed with IL-2 RNA. Ratios of the peak areas of hybrids to all the peakareas in the fluorescence chromatogram, estimated for each probe, weresummarized and shown in Table 6.

TABLE 6 Ratio of Name of Probe Base Sequence Hybrid (%) IL-2 228–5′-GTAAAACTTAAATGT-3′ 0.1 242 (D) IL-2 243– 5′-GGCCTTCTTGGGCAT-3′ 17.5257 (D) IL-2 198– 5′-TTTGGGATTCTTGTA-3′ 15.7 212 (D) IL-2 213–5′-GAGCATCCTGGTGAG-3′ 25.2 227 (D) IL-2 77–91 (D) 5′-GCAAGACTTAGTGCA-3′0.5 IL-2 92– 5′-CTGTTTGTGACAAGT-3′ 18.3 106 (D) IL-2 287–5′-GGTTTGAGTTCTTCT-3′ 13.3 301 (D) IL-2 302– 5′-AGCACTTCCTCCAGA-3′ 6.2316 (D) IL-2 342– 5′-CCTGGGTCTTAAGTG-3′ 22.8 356 (D) IL-2 357–5′-ATTGCTGATTAAGTC-3′ 27.3 371 (D)

From the results shown in Table 6, it was found that IL-2 213–227(D),IL-2 342–356(D), and IL-2 357–371(D) were hybridized to the target RNAwith relatively high efficiency. In the results of (3A), the combinationof IL-2 342–356(D) and IL-2 357–371(A) caused the largest changes influorescence spectra as a pair of a donor probe and an acceptor probewhen the probes were mixed with IL-2 RNA. Therefore, the results of (4A)are well consistent with those of (3A).

(4B) Measurement of Hybridization Efficiency of Probes to Human IL-4 RNAby HPLC

Each donor probe (3 pmol) wherein an oligo DNA corresponding SEQ ID NO:11–20 was labeled with Bodipy493/503 was mixed with an equimolar amountof human IL-4 RNA, synthesized in (2B) in 10 μl of 1×SSC solution (150mM sodium chloride, 17 mM sodium citrate, pH 7.0) and the mixture wasallowed to stand at room temperature for 15 minutes. Then, hybridsconsisting of human IL-4 RNA and probes were separated from free probesby high performance liquid chromatography (HPLC) using differences inretention time under the following conditions: retention time is about4–5 minutes for free probe and about 7.5 minutes for hybrid in the HPLCcondition below. The conditions for separation and the method fordetermining hybridization efficiency are the same as those described in(4A).

FIGS. 15, 16, 17 and 18 show HPLC chromatograms when IL-4 119–133 (D),IL-4 134–148(D), IL-4 134–148(D), IL-4 265–279(D), and IL-4 280–294(D)were mixed with IL-4 RNA, respectively. Ratios based on the peak areasof hybrids to all the peak areas in the fluorescence chromatogram,estimated for each probe, were summarized and shown in Table 7.

TABLE 7 Hybridized Ratio of Position Base Sequence Hybrid (%) IL-4 70–84(D) 5′-CAGTTGGGAGGTGAG-3′ 68.6 IL-4 85–99 (D) 5′-GAACAGAGGGGGAAG-3′ 53.5IL-4 119– 5′-CGTGGACAAAGTTGC-3′ 4.2 133 (D) IL-4 134–5′-TATCGCACTTGTGTC-3′ 22.6 148 (D) IL-4 176– 5′-CTGTGAGGCTGTTCA-3′ 23.7190 (D) IL-4 191– 5′-ACAGAGTCTTCTGCT-3′ 1.5 205 (D) IL-4 265–5′-AGCCCTGCAGAAGGT-3′ 15.6 279 (D) IL-4 280– 5′-CCGGAGCACAGTCGC-3′ 46.6294 (D) IL-4 376– 5′-CCGTTTCAGGAATCG-3′ 23.1 390 (D) IL-4 391–5′-GAGGTTCCTGTCGAG-3′ 4.0 405 (D)

From the results shown in Table 7, it was found that IL-4 265–279(D) andIL-4 280–294(D) were hybridized to the target RNA with relatively highefficiency. In the results of (3B), the combination of IL-4 265–279(D)and IL-4 280–294(A) caused the largest changes in fluorescence spectraas a pair of a donor probe and an acceptor probe when the probes weremixed with IL-4 RNA. Therefore, the results of (4B) are well consistentwith those of (3B).

(5) Induction of IL-2 Gene Expression in Human T-cell Leukemia StrainCells Jurkat E6-1

To Jurkat E6-1 cells with a cell density of 1×10⁶/ml was added 0.5 mg/ml(final concentration) of anti-CD3 antibody (Immunotech Inc.), 0.5 mg/mlanti-CD28 antibody (Immunotech Inc.), and 10 nM PMA (Sigma Inc.), andthey were cultured for 3 days (24 hours) at 37° C. in the presence of 5%CO₂.

(6) Measurement of the Production Amount of IL-2 Protein Molecules

If a large amount of IL-2 molecules is produced and liberated intoculture supernatant in response to the induction of IL-2 gene expressionin (5), it is plausible that IL-2 mRNA is actively synthesized in thecells. Thus, in order to confirm IL-2 gene expression, culturesupernatant of Jurkat E6-1 cells treated with IL-2 expression-inducingagents (in some cases, hereinafter called IL-2 expression-induced cells)which had been treated as described in (5) was collected; the amount ofIL-2 (pg/ml/10⁷ cells) in the supernatant was determined by the ELISAsandwich method (which will be described below) using Humaninterleukin-2 measurement kit (Japan Immunoresearch Laboratories Co.,Ltd.) and the amounts of IL-2 in the supernatant of the treated cellswere compared with those for untreated cells (in some cases, hereinaftercalled IL-2 expression-uninduced cells).

Wells in a 96-well plate (antibody plate) on which anti-human IL-2monoclonal antibodies were immobilized were washed with washing solutiontwice, and 150 μl of buffer solution was added to each well to be used.To each well was added 50 μl of the supernatant of culture medium orpurified IL-2 (0–1,600 pg/ml, standard human IL-2 protein in Human IL-2measurement kit), they were incubated at 37° C. overnight. The reactionsolution in each well was removed, and the well was washed with washingsolution three times. The first antibody (anti-human IL-2 rabbit serum)solution was added at 100 μl/well, and incubated at room temperature for2 hours. The antibody solution in each well was removed, followed bywashing with the washing solution three times.

The second antibody (peroxidase labeled anti-rabbit IgG antibodies)solution was added at 100 μl/well, and incubated at room temperature for2 hours. The antibody solution in each well was removed. After the wellwas washed with washing solution three times, and fully dried. Aperoxidase substrate solution, o-phenylenediamine dissolved in 0.015%hydrogen peroxide was added at 100 μl/well, and was allowed to react atroom temperature for 10–20 minutes. 100 μl of 1 N H₂SO₄ was added toeach well to stop the reaction. Absorbance at 492 nm in each well wasmeasured using a microplate reader. IL-2 in the culture supernatant wasquantified based on the calibration curve created from the values ofabsorbance of standard IL-2.

6157±168 (pg/ml/10⁷ cells) of IL-2 were detected in the culture mediumof IL-2 expression-induced cells, while the amount of IL-2 was less thanthe detectable range for IL-2 expression-uninduced cells (<0.1 pg/ml/10⁷cells)

(7) Measurement of Amounts of IL-2 Gene Expression

For experimental materials, this experiment required purified IL-2 RNAas a reference sample, the total RNA extracted from Jurkat B6-1 cells asa measurement sample, and ribonucleic acid probe of IL-2 (RNA probe forIL-2) labeled with digoxigenin for the detection of IL-2 RNA or IL-2mRNA. They were obtained using the methods in the following (a)–(c).

(a) Standard IL-2 RNA

Human IL-2 RNA (1 μg/μl) synthesized in (2) was diluted by 10⁴, 10⁵,10⁶, and 10⁷ times with 1×dilution buffer (RNase-free sterile distilledwater: 20×SSC: formamide=5:3:1). The diluted RNA solutions were heatedat 68° C. for 10 minutes, then quenched on ice, and then used forblotting.

(b) Total RNA of Jurkat E6-1 Cells

Total RNA of Jurkat E6-1 cell was extracted using an RNeasy kit (QIAGENInc.). Under the conditions shown in (5), cells treated with the IL-2expression-inducing agent for 0, 24, 48, 72, and 96 hours (0.8–1.2×10⁷cells) were recovered as precipitates by centrifugation at 1,500 rpm for5 minutes. The cells were suspended in 1,000 μl of homogenization buffercontaining 10 μl of β-mercaptethanol and were denatured sufficiently byrepeating a manipulation of suction/emission with a syringe in18-gauges. To the homogenate was added 1,000 μl of 70% ethanol, then itwas applied to a column for RNA absorption, centrifuged at 4,000×g for 5minutes, and then the column was washed once by centrifugation after theaddition of washing buffer.

RNase-free sterile distilled water was added to the column to elute theabsorbed RNA. To the eluted RNA solution was added 0.1 times volume of 4M sodium acetate and an equal volume of isopropanol. Then RNA wasrecovered as a precipitate by centrifugation at 15,000×g for 15 minutesand. After the RNA was dissolved in the RNase-free sterile distilledwater, it was diluted with an equal volume of 2× dilution buffer(RNase-free sterile distilled water: 20×SSC: formamide=1:6:2), heated at68° C. for 10 minutes and then quenched quickly.

(c) Digoxigenin (DIG)-Labeled RNA Probe for IL-2 RNA

DIG-labeled RNA probe for IL-2 RNA was synthesized using a DIG RNALabeling kit (Boehringer Mannheim Inc.). 10 μg of human IL-2 cDNArecombinant plasmid DNA (pTCGF#2), linearized by EcoRI digestion, waspurified by ethanol precipitation. After removing the enzyme proteindenatured with phenol/chloroform, the purified DNA was used as atemplate for RNA probe synthesis. The template DNA (5 μg) and 1.8 mMATP, 0.9 mM CTP, 0.7 mM GTP, 1.1 mM UTP, and 0.58 mM UTP (DIG-labeled)were mixed in the presence of T7 RNA polymerase. The mixture wasincubated at 37° C. for 2 hours. Then, DNase I solution was added andreacted for 10 minutes to degrade the template DNA. To the reactionsolution were added 0.1 times volume of 5 M sodium acetate and an equalvolume of isopropanol, and the synthesized RNA was recovered bycentrifugation at 15,000×g for 15minuts. The RNA was then dissolved inRNase-free sterile distilled water.

(d) Measurement of Amounts of IL-2 Gene Expression

Using the materials obtained from (a)–(c), the amount of IL-2 geneexpression was measured. The cellular total RNA solution and standardIL-2 RNA solution were dotted to a nylon membrane, and washed with 5×SSCtwice. Then, the RNA on the nylon membrane was fixed using aUV-Crosslinker (Biorad Inc.). The nylon membrane, prehybridizationbuffer (5×SSC, 5% SDS, 50 mM sodium phosphate (pH 7.0), 50% formamide,2% Blocking Reagent (Boehringer Mannheim Inc.), and 1% N-laurylsarcosinate were enclosed into a HybriBag (Iuchi Inc., Hot waterresistant bag:L) and incubated at 68° C. for one hour.

DIG-labeled probe for IL-2 RNA was diluted with a prehybridizationbuffer to give a final concentration of 100 ng/ml, boiled for 10 minutesand then quenched to prepare hybridization solution. Prehybridizationsolution in HybriBag was replaced with the hybridization solution, andhybridization was carried out at 68° C. overnight. The nylon membranewas washed with 2× Washing solution (2×SSC, 0.1% SDS) twice for 5minutes each, and with 0.2× Washing solution (0.2×SSC, 0.1% SDS) at 68°C. for 15 minutes each. After washing the nylon membrane with Buffer I(100 mM maleic acid, 150 M Nacl (pH 7.5)) for one minute, the nylonmembrane was incubated with Buffer II (Blocking Reagent (BoehringerMannheim Inc.) was diluted to 1% with buffer 1).

The amounts of hybrid formed with DIG-labeled probes and IL-2 RNA, wereestimated by chemiluminescence emitted from the hybrids using DIGLuminescent Detection kit (Boehringer Mannheim Inc.) as follows. Thenylon membrane was treated with alkalinephosphatase-labeled anti-DIGantibodies (150 mU/ml in Buffer II solution), at room temperature for 1hour. Then the nylon membrane was washed with Buffer I twice for 15minutes each, and packed into a bag (LIFETECHNOLOGIES Inc., Photogenedevelopment folder) with 250 μM of substrate solution which had beenobtained by diluting CSPD (disodium3-(4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate) with Buffer III(0.1 M Tris, pH 9,7, 0,1 M NaCl, 0.05MgCl₂). The number of photons emitted from the enzymatically degradedsubstrate was counted with ARGUS 50 (Hamamatsu Photonics). A calibrationcurve indicating the relation between the number of photon and theamount of standard IL-2 RNA was created. Based on the curve, the amountof IL-2 mRNA out of total cellular RNA in the cell was determined. Fromthe amount of IL-2 mRNA (mol) obtained and the number of cells used forextraction of the total cellular RNA, the number of IL-2 mRNA moleculesper single cell was determined (FIG. 19).

As shown in FIG. 9, the numbers of IL-2 mRNA molecules in the singlecell where the cells were treated for inducing IL-2 expression for 0,24, 48, 72, and 96 hours were <0.29, (0.76±0.17)×10⁴, (1.11±0.40)×10⁴,(1.22±0.67)×10⁴, and (1.20±0.28)×10⁴, respectively. The cells treatedfor 72 hours (3 days) were found to contain the maximum number of IL-2mRNA. In addition, the contents of extracellular IL-2 (pg/ml/10⁷ cells)of the cells treated for the above described periods were <0.32,1,032±25, 2,433±533, 2,688±194, and 2,531±283, respectively. Therefore,it was suggested that more IL-2 molecules had been secreted from thecells with higher efficiency in IL-2 gene expression.

(8) Intracellular Hybridization Between Each Probe and IL-2 mRNA inHuman T-Cell Leukemia Strain Jurkat E6-1 Cells Induced the Expression ofIL-2 Gene

For IL-2 expression-induced cells the IL-2 gene expression of which wasinduced by treatment with anti-CD3 antibodies, anti-CD28 antibodies, andPMA for 3 days as described in (5), detection of the hybridizationbetween intracellular IL-2 mRNA and each IL-2 probe was performed by IST(In Situ Transcription) as follows.

The IL-2 expression-induced and -uninduced Jurkat E6-1 cells (5×10⁵cells/ml) were washed with PBS(−) three times, and suspended with 1 mlof PBS(−), and the suspension was mounted on 12 mm of a cover glass(poly-L-lysine was coated on the bottom) to prepare a monolayer ofcells. The cells were exposed to 0.5% Triton-X100 solution for 90seconds at room temperature to permeabilize the cells. After thepermeabilized cells were quickly washed with PBS(−), 10 μM (finalconcentration) of probes corresponding to SEQ ID NO: 1–10 in Table 1(unlabeled with dye), oligo dT (deoxythymidine oligonucleotide,unlabeled with dye) or oligo dA (deoxyadenine oligonucleotide, unlabeledwith dye) were added, and incubated for one hour at room temperature.The cells were washed with PBS(−) quickly, and fixed with 4%paraformaldehyde solution at room temperature for 15 minutes.

The cells were washed with 1×SSC three times; 1 mM deoxyribonucleotidesolution (Boehringer Mannheim Inc.) containing 0.35 mM of DIG(digoxigenin)—labeled dUTP and 1 u/μl reverse transcriptase (Toyobo,Inc.) were added to the cells; and they were incubated for 2 hours at30° C. The cells were washed with 1×SSC three times, and treated withBlocking buffer [Blocking Reagent (Boehringer Mannheim Inc.) dissolvedin maleic acid buffer solution so that its ratio would be equal to 1(w/v) %]. The cells were washed with maleic acid buffer solution threetimes. FITC (Fluorescein-isothiocyanate) labeled anti-DIG antibody(which was diluted with Blocking buffer up to 1 μg/ml) was added to thecells and then the cells were incubated for 30 minutes at roomtemperature. The cover glass was washed with PBS(−) three times,observed under a fluorescence microscope, and the total fluorescenceintensity in the visual field (relative value) was measured.

FIGS. 20 and 21 show the fluorescence micrographs obtained. FIG. 20shows a fluorescence micrographs where the hybrids formed between all ofthe cellular mRNA and oligo dT were fluorescently detected in the fixedIL-2 expression-induced cells and -uninduced cells. FIG. 20 also showsthe control experiments in cases where oligo-dA was added instead ofoligo dT, or where both DIG-labeled dUTP and reverse transcriptaserequired for fluorescent labeled complex were not added. FIG. 21 showsfluorescence micrographs in which hybrids, formed between IL-2 mRNA andeach probe (fluorescently unlabeled), were fluorescently labeled in thefixed IL-2 expression-induced and -uninduced. In FIG. 21, twofluorescence images for each probe are shown for IL-2 expression-inducedand -uninduced cells. FIG. 22 shows the normalized intensity (%) of eachprobe out of the fluorescence intensity obtained from the addition ofoligo dT to the cells, the relative fluorescent intensity per cell wasworked out from the fluorescence intensities (mean value±SE) divided bythe number of cells in the corresponding phase contrast micrograph.

IL-2 342–356 and IL-2 357–371 were shown to hybridize with intracellularIL-2 mRNA most efficiently. As these two probes are complementary to anadjacent site on IL-2 mRNA, if one of the two probes is labeled with anfluorescent energy donor dye and the other is labeled with anfluorescent energy acceptor dye, FRET fluorescence caused byintracellular hybridization could be detected specifically.

(9) Intracellular Hybridization (ISH) of Donor Probes and AcceptorProbes with IL-2 mRNA in IL-2 Expression-Induced Cells

In the experimental results by IST in (8), the probes were introducedinto almost all the cells uniformly in a visual field of a fluorescencemicroscope. The method for introducing probes in (8) was also used whenfluorescent labeled probes of donors and acceptors were introduced intocells. Hybridization between these probes and intracellular IL-2 mRNAwas detected as FRET fluorescence. Various sets of probes were examined.Since this method modifies the IST method, it is called ISH (In SituHybridization).

A solution of 2 μM (final concentration) of a donor probe labeled withBodipy 493/503 and an acceptor probe labeled with XRITC was added toIL-2 expression-induced cells as well as to IL-2 expression-uninducedcells, both of which were provided with material permeability in thecellular transmembranes as described in (8), and they were incubated forone hour at room temperature. As sets of donor probes and acceptorprobes, IL-2 228–242(D) and IL-2 243–257(A), IL-2 198–212(D) and IL-2213–227(A), IL-2 77–91(D) and IL-2 92–106(A), IL-2 287–301(D) and IL-2302–316(A), and IL-2 342–356(D) and IL-2 357–371(A) were used. After thecells were washed with PBS(−), the cells were fixed with 4%paraformaldehyde solution for 15 minutes at room temperature. The coverglass was washed with PBS(−) three times before fluorescence microscopy.Three kinds of fluorescence images were obtained as follows. These arethe fluorescence of A emitted from the cells when the excitation lightof A (energy acceptor dye) was irradiated to the cells (in some cases,hereinafter called A/A image), the fluorescence of D emitted from thecells when the excitation light of D (energy donor dye) was irradiatedto the cells (hereinafter, sometimes called D/D image), and thefluorescence of A emitted from the cells when the excitation light of Dwas irradiated to the cells (in some cases, hereinafter called D/Aimage). The D/A image represents fluorescence caused by FRET. Themaximum intensity of FRET fluorescence was obtained when IL-2 342–356(D)and was used as a donor probe and IL-2 357–371(A) as an acceptor probe.These fluorescence micrographs are shown in FIG. 23. In FIG. 23, twosets of D/A, A/A, and D/D images for each pair of probes are shown forIL-2 expression-induced and -uninduced cells. In this figure, the imagesin which probes were not introduced are also shown as a controlexperiment.

The total fluorescence intensity in a D/A image, acceptor fluorescenceof FRET with donor excitation, and a A/A image, acceptor fluorescencewith acceptor excitation, were obtained. Then, the average fluorescenceintensity value per cell in a D/A and A/A image was estimated as thetotal fluorescence intensity divided by the number of cells in acorresponding image. In order to evaluate the intracellularhybridization efficiency, the average D/A value per cell/the average A/Avalue per cell, representing the ratio of the hybridized acceptor probesto IL-2 mRNA to the total probes, were obtained. The value of(D/A)/(A/A) (%) for each pair of probes is shown in FIG. 24. From theresults in (8) and (9), it is suggested that IL-2 342–356(D) and IL-2357–371(A) hybridize to the target mRNA in the cells individually aswell as adjacently.

(10) Intracellular Hybridization when Donor Probes and Acceptor Probeswere Introduced into Live IL-2 Expression-Induced Cells

Throughout the results of (3A)-(9), IL-2 342–356(D) and IL-2 357–371(A)were selected as probes to detect IL-2 mRNA in live cells. Thesefluorescently labeled probes were introduced into live cells expressingIL-2 genes; and the hybridization was specifically measured based on thechanges in FRET fluorescence.

As described in (5), IL-2 expression-induced cells which were preparedby the treatment with anti-CD3 antibodies, anti-CD28 antibodies and PMAfor three days, and -uninduced cells, were washed with ice-cold PBS(−)twice, and suspended in PBS(−) to 1×10⁷ cells/ml. Then, 0.9 ml of thecell suspension was transferred into a cuvette for electroporation; and5.4 nmol (final concentration; 6.0 μM) of Bodipy493/503-labeled donorprobe IL-2 342–356(D) and 5.3 nmol (final concentration; 5.86 μM) ofXRITC-labeled acceptor probe Il-2 357–371(A) were added; and the cellswere pulsed at 250 V, 975 μF. After the cell suspension was filteredthrough 70 μm of Cell Strainer (Falcon) and centrifuged mildly, thecells were resuspended with PBS(−). Further the suspended solution wasfiltered through 40 μm of Cell Strainer (Falcon); the re-passed solutionwas centrifuged and resuspended to remove debris including dead cells asmuch as possible; and the suspended solution was observed under afluorescence microscope.

The results were shown in FIG. 25. FIG. 25 shows A/A, D/D, and D/Aimages, and the corresponding phase contrast image of IL-2expression-induced and -uninduced cells. In FIG. 25, two sets of A/A,D/D, D/A images, and the corresponding phase contrast images for eachpair of probes were shown. One to three cells out of 20–22 cells wereD/A-positive cells in the visual field for IL-2 expression-inducedcells, suggesting that the donor probe and the acceptor probe adjacentlyhybridize to IL-2 mRNA. On the other hand, no D/A-positive cells wereobserved for IL-2 expression-uninduced cells.

(11) Selective Separation of Cells Which-Have Expressed IL-2 Genes byFlow Cytometry

Utilizing the differenciated intensities of FRET-fluorescence betweenIL-2 expression-induced and -uninduced cells based on the specifichybrid formation among the donor probe, the acceptor probe, and IL-2mRNA, it was attempted to separate IL-2 expressing cells fromnon-expressing cells as follows.

The cell suspensions of IL-2 expression-induced cells prepared asdescribed in (9) and those of -uninduced cells were mixed with theratios of 100:0, 0:100, 50:50, and 20:80, respectively. Then, 0.9 ml ofthe mixture was put into a cuvette; 16.2 nM (final concentration; 18.0μM) of Bodipy493/503-labeled donor probe IL-2 342–356(D) and 14.7 nmol(final concentration; 16.4 μM) of Cy5-labeled acceptor probe IL-2357–371(A) were added. The mixture with the probes was pulsed asdescribed in (9). Live cells were collected and were applied to a flowcytometer, an experimental device for flow cytometry (FACSCalibur,BECTON DICKINSON Inc.).

At a position in flow path, the excitation light for an energy donorfluorescence dye (Bodipy493/503) was irradiated to the cells. Relativefluorescence intensity emitted from acceptor fluorescence dye (Cy5),representing FRET fluorescence based on the hybridization was measuredtogether with relative intensity of Bodipy493/503. Each cell was plottedas a dot in a diagram with the X-axis, the intensity of Bodipy493/503(FL1-Height) and Y-axis, that of Cy5 (FL3-Height). Among these plots, agroup of dots with the highest value of FL3-Height representing a groupof fluorescing cells based on hybridization, was designated as R2. Onthe other hand, a group of dots, representing the cell size (FSC-Height;forward-scattering light) as well as the complexity in theintrastructure (SSC-Height; side-scattering light) as typical humanlymphoid cells was designated as R1 according to the reference value(FACSCalibur Training Manual, BECTON DICKINSON).

The obtained dot-plots were shown in FIGS. 26–33. FIG. 26 shows a dotplot for FSC-Height and SSC-Height when the mixing ratio of IL-2expression-induced cells to -uninduced cells was 100 to 0. FIG. 27 showsa dot plot of the cells with the same mixing ratio based on FL1-Heightand FL3-Height as in FIG. 26. FIG. 28 shows a dot plot of the cellsbased on FSC-Height and SSC-Height when the mixing ratio of IL-2expression-induced cells to -uninduced cells was 0 to 100. FIG. 29 showsa dot plot of the cells based on the FL1-Height and FL3-Height with thesame mixing ratio as in FIG. 28. FIG. 30 shows a dot plot of the cellsbased on FSC-Height and SSC-Height when the mixing ratio of IL-2expression-induced cells to -uninduced cells was 50 to 50. FIG. 31 showsa dot plot of the cells based on FL1-Height and FL3-Height with the sameratio as in FIG. 30. FIG. 32 shows a dot plot of the cells based onFSC-Height and SSC-Height when the mixing ratio of IL-2expression-induced cells to -uninduced cells was 20 to 80. FIG. 33 showsa dot plot of the cells based on FL1-Height and FL3-Height with the samemixing ratio as in FIG. 32.

A cell group belonging to both R1 and R2 was collected by a cell sortingfunction (a cell sorter). Some of the collected cell group was appliedto FACSCalibur and detected as similar dot-plots in order to confirmthat the group was still fluorescently labeled as desired. The obtaineddot-plots were shown in FIGS. 34–39. FIG. 34 shows a dot plot forFSC-Height and SSC-Height when the mixing ratio of IL-2expression-induced cells to -uninduced cells was 100 to 0. FIG. 35 showsa dot plot of the cells based on FL1-Height and FL3-Height with the samemixing ratio as in FIG. 34. FIG. 36 shows a dot plot for FSC-Height andSSC-Height when the mixing ratio of IL-2 expression-induced cells to-uninduced cells was 50 to 50. FIG. 37 shows a dot plot of the cellsbased on FL1-Height and FL3-Height with the same mixing ratio as in FIG.36. FIG. 38 shows a dot plot for FSC-Height and SSC-Height when themixing ratio of IL-2 expression-induced cells to -uninduced cells was 20to 80. FIG. 39 shows a dot plot of the cells based on FL1-Height andFL3-Height with the same mixing ratio as in FIG. 38.

The comparison of FIG. 27, FIG. 31, and FIG. 33 revealed that theproportion of dots representing the cell group belonging to R2 to theentire number of dots decreased in relation to the decrease in themixing ratio of IL-2 expression-induced cells from 100, 50 to 20%, whilethe value of FL3-Height was totally background level regarding IL-2expression-uninduced cells even when the same donor and acceptor probeswere introduced to the cells (FIG. 29). As most of the sorted-out cells,selectively separated and collected cells, belonged to both R1 and R2,IL-2 expression-induced cells were found to be collected as the livecells emitting considerable FRET-fluorescence (see FIGS. 34–39).

(12) Comparison of Cell Groups Before and After Flow Cytometry byFluorescence Microscopy

Some of the cells before and after flow cytometry were transferred toglass-bottomed dishes, and ratios of fluorescing cells of D/A, A/A, andD/D of the cells in the entire visual field were examined.

The results were shown in FIGS. 40–46. FIGS. 40, 42, 44, and 46 showimages of the cells before flow cytometry; fluorescence images ofacceptor dyes based on FRET representing hybrid formation among IL-2mRNA, donor probes and acceptor probes (D/A image), fluorescence imagesof donor dyes by the donor-excitation representing the presence of donorprobes in the cells (D/D image), fluorescence images of acceptor dyes bythe acceptor-excitation representing the presence of acceptor probes inthe cells (A/A image), and the corresponding phase contrast images.FIGS. 41, 43 and 45 show images of the cells after flow cytometry, thecells selectively collected by the cell sorting function. The A/A, D/A,and D/D fluorescence images and the corresponding phase contrast imagesare shown in these figures as in FIGS. 40, 42, 44 and 46. In FIGS.40–46, arrows indicating some cells are shown to align the positions ofcells between the fluorescence images and the phase contrast images.

Cells in FIGS. 40 and 41 were the mixture of IL-2 expression-induced and-uninduced live cells with the ratio of 100 to 0; cells in FIGS. 42 and43 were those with the ratio of 50 to 50. Cell groups in FIGS. 34 and 35were those with the ratio of 20 to 80; and cell groups in FIG. 46 werethose with the ratio of 0 to 100.

In FIG. 40, the description of 20 cells in the phase contrast micrographmeans that there were 20 cells in the entire visual field. Thedescriptions of 5 cells, 4 cells and 7 cells in A/A, D/A and D/D imagesrepresent that the numbers of the cells emitting A/A-, D/A-, andD/D-fluorescence were 5, 4, and 7, respectively.

In FIG. 42, the descriptions of 20 cells, 3 cells, 1 cell and 10 cellsmean that the number of the cells in the entire visual field was 20 andthe numbers of cells emitting A/A-, D/A-, and D/D-fluorescence were 3,1, and 10 respectively. In FIG. 44, the descriptions of 36 cells, 7cells, 1 cell and 20 cells mean that the number of the cells in theentire visual field was 36 and the numbers of cells emitting A/A-, D/A-,and D/D-fluorescence were 7, 1, and 20, respectively. In FIG. 46 whereIL-2 expression-uninduced cells are contained, the descriptions of 21cells, 2 cells, 0 cell and 3 cells mean that the number of the cells inthe entire field was 21 and the numbers of cells emitting A/A-, D/A-,and D/D-fluorescence were 2, 0, and 3 respectively.

Thus, as the ratio of IL-2 expression-induced cells was decreased from100, 50, to 20%, it was found that the ratio of D/A-fluorescent cellspossessing fluorescently labeled IL-2 mRNA to the cells in the entirevisual field decreased.

FIG. 41 shows fluorescence images of the cells selectively separatedfrom the mixture of IL-2 expression-induced and -uninduced live cellswith the ratio of 100 to 0. The description of 7 cells in the phasecontrast micrograph means that 7 cells exist in the entire visualfields. The descriptions of 7 cells, 7 cells, and 7 cells in the A/A,D/A and D/D images represent that all 7 cells in the field were emittingA/A-, D/A- and D/D-fluorescence.

In FIG. 43, the descriptions of 6 cells in the phase contrast image andin all the A/A, D/A and D/D images represent that the number of thecells in the entire field was 6 and that all the cells were emittingA/A-, D/A-, and D/D-fluorescence. Similarly in FIG. 45, all thedescriptions of 5 cells in the phase contrast image and in all the A/A,D/A and D/D images represent the number of the cells in the entire fieldwas 5 and that all the cells were emitting A/A-, D/A-, andD/D-fluorescence.

The comparison between FIGS. 40 and 41, between FIGS. 42 and 43, andbetween FIGS. 44 and 45 revealed that only the cells in which IL-2 mRNAwas fluorescently labeled could be selectively separated by the cellsorting function of flow cytometry (a cell sorter).

(13) Comparison Between the Cells Before and After Flow Cytometry by InSitu Hybridization

Some of the cells before and after flow cytometry were transferred to aglass-bottomed dish, and fixed with 4% paraformaldehyde/PBS (pH7.4) atroom temperature for 30 minutes. The ratio of cells carrying IL-2 mRNA(IL-2 mRNA (+)) in the entire visual field was determined by fluorescentin situ Hybridization. First, in this FISH, in order to prevent higherbackground caused by RNA probes remaining in the cell after the wash-outprocedure, IL-2 RNA probes, which were obtained by the fragmentation ofa full-length anti-sense IL-2 RNA synthesized according to the method in(7) (c), were used for hybridization experiments.

A full-length anti-sense IL-2 RNA, 10 μg, were dissolved in 100 μl ofalkaline solution (42 mM NaHCO₃, 63 mM Na₂CO₃, 5 mM DTT), incubated at60° C. for 10–15 minutes. Then, 10 μl of 3 M sodium acetate and 350 μlof ethanol were added to precipitate the RNA probes. After allowing tostand at −20° C. for 30 minutes, they were and centrifuged at 16 krpmfor 20 minutes. The obtained precipitates were washed with 70% ethanoland dried. The precipitates were dissolved in 50 μl of RNase-freesterile distilled water to prepare an alkaline denatured RNA probesolution for IL-2 RNA.

The cells fixed at the bottom of the dish were washed with PBS(−) threetimes and treated with 0.1% Triton X-100/PBS solution at roomtemperature for 5 minutes to permeabilize the cells, and thepermeabilized cells were washed with PBS(−) three times and treated with0.2N HCl at room temperature for 10 minutes. After washing the monolayercells with PBS(−), they were incubated with 1 μg/ml of proteinase K/PBSsolution for 5 minutes at 37° C. After the monolayer cells were washedwith PBS(−), they was fixed again with 4% of paraformaldehyde/PBS (pH7.4) for 30 minutes. The fixed cells were washed twice with 2 mg/ml ofglycine/PBS for 15 minutes, and treated with 50% deionizedformamide/2×SSC solution (solution A, described hereunder) for 30minutes; the hybridization solution (50% deionized formamide, 5×denhardt, 2×SSC, alkaline denatured probes for IL-2 RNA (1 μg/ml)) wasprepared, denatured at 90° C. for 10 minutes, and then the monolayercells were ice-cooled. Adding 100 μl of the hybridization solution tothe cells, they were incubated at 45° C. overnight.

The monolayer cells after the hybridization were washed twice withsolution A for 5 minutes at 45° C., then washed with solution B (10 MTris. HCl(pH8.5), 500 mM NaCl) for 5 minutes, and treated with 20 μg/mlof RNase A/solution B (pretreated at 90° C. for 10 minutes) at 37° C.for 20 minutes. They were washed with solutions A at 45° C. for 30minutes and C (50% of deionized formamide/1×SSC) at 45° C. for 30minutes and with solution C at room temperature for 20 minutes. Afterwashing with Buffer 1 [100 mM maleic acid, 150 mM NaCl (pH7.5)] twicefor 5 minute, they were treated with Buffer 2 [1% BlockingReagent(Boehringer Mannheim Inc.) in Buffer 1] at room temperature for20 minutes.

After the monolayer cells were washed with Buffer 1 twice, FITC-labeledanti-DIG antibodies (Fab, diluted with Buffer 2 by 100 times, proteinlevel: about 1 μg/ml) was added, incubated for at least 30 minutes,washed with PBS(−) three times. The cells were observed under afluorescence microscope; and the ratio of cells carrying IL-2 mRNA tothe total cells in the visual field was determined.

FIGS. 47 and 48 show fluorescence micrographs when the mixing ratio ofIL-2 expression-induced cells to -uninduced cells was 100 to 0. FIG. 47shows micrographs before flow cytometry. FIG. 48 shows micrographs afterflow cytometry. FIG. 49 shows fluorescence micrographs before flowcytometry when the mixing ratio of IL-2 expression-induced cells to-uninduced cells was 0 to 100. FIGS. 50 and 51 show fluorescencemicrographs when the mixing ratio of IL-2 expression-induced cells to-uninduced cells was 50 to 50. FIG. 50 shows micrographs before flowcytometry and FIG. 51 shows those after flow cytometry. FIGS. 52 and 53show fluorescence micrographs when the mixing ratio of IL-2expression-induced cells to -uninduced cell was 20 to 80. FIG. 52 showsfluorescence micrographs before flow cytometer, and FIG. 53 shows thoseafter flow cytometry.

The figures in the micrographs represent the numbers of fluorescingcells per the number of total cells in the entire visual field. In FIG.47, “48/48” represents that all of the 48 cells were fluorescing cellsin the image, suggesting that all the cells were IL-2 mRNA carryingcells. On the contrary, “0/32” in FIG. 49 represents that there were noIL-2 mRNA carrying cells out of 32 cells. In FIG. 50, “18/35” representsthat 18 cells possess IL-2 mRNA out of 35 cells. In FIG. 52, “8/39”represents that 8 cells possess IL-2 mRNA out of 39 cells. These resultsare well consistent with the fact that IL-2 expression-induced and-uninduced cells were mixed with the ratios of 50 to 50 in FIGS. 50 and20 to 80 in FIG. 52. On the other hand, the figures in FIG. 48, FIG. 51,and FIG. 53 were 9/9, 7/7, and 8/8, respectively, suggesting that IL-2mRNA carrying cells were condensed from 20–50% to 100% throughout flowcytometry.

(14) Separation Method Based on Fluorescence Intensities of Live CellsExpressing Specific Genes

Table 8 is a summarized result of (11)–(13) to show the effect of thisseparation method utilizing the differenciated intensities based on thefluorescent labeling of IL-2 mRNA. The cells carrying IL-2 mRNA whichwere condensed from 20–50% to 100% by the separation method utilizingthe difference in fluorescence intensities. That is, all the live cellscarrying IL-2 mRNA were selectively separated from the live cell groupcontaining, IL-2 mRNA carrying cells.

TABLE 8 Flow Cytometry Before After FRET Cells FRET Cells Mixing Ratios(D/A) carrying (D/A) carrying IL-2 expression Positive IL-2 mRNAPositive IL-2 mRNA Induced uninduced Cells (%) (%) Cells (%) (%) 100 020 100 100 100 0 100 0 0 — — 50 50 5 51.4 100 100 20 80 2.8 20.5 100 100(15) Separation of Lymphocytes from Human Peripheral Blood

After sampling 200 ml of blood from a healthy adult and adding heparinthereto at a final concentration of 10 U/ml, it was mixed with atwo-fold volume of 3% dextrin in PBS (phosphate buffered saline) in a 50ml centrifugation tube (Falcon 2070), and the mixture was allowed tostand at room temperature for 15 minutes to precipitate erythrocytes.21.5 ml of the supernatant was gently superposed onto a 15 ml of Ficollpack (Pharmacia) and subjected to centrifugation at 490×g (1,600 rpm)for 30 minutes. The lymphocyte layer observed as white suspended matterin the supernatant was collected with a pipette. After mixing thelymphocyte layer with a three-fold volume of HBSS (Hanks' Balanced SaltSolution, GIBCO BRL) in a 50 ml centrifugation tube (Falcon 2070) andcentrifuging at 1,200 rpm for 10 minutes, the lymphocyte precipitate waswashed twice with 30 ml of PBS(−) and suspended to a cell density ofabout 2.5×10⁷ cells/ml.

(16) Separation and Fluorescent Antibody Staining of Helper T Cells(CD4+ cells) from Peripheral Blood Lymphocytes

Separation of CD4+ cells from peripheral blood lymphocytes was performedusing Human CD4+ cell Recovery Column Kit (CEDARLANE Laboratories, Ltd.)was used for separation of the CD4+ cells, by a separation procedureaccording to the protocol provided with the kit.

A CD4+ cell separating column (CEDARLANE) was set in a column stand andthe glass beads in the column were equilibrated with 15 ml of PBS(−),with only a slight amount of the PBS(−) remained above the beads. Tocoat these beads with goat anti-human IgG (H+L) and goat anti-mouse IgG(H+L), the powder of Column Reagent (CEDARLANE) was dissolved in 1–1.5ml of PBS(−), and applied to the column and allowed to flow till only aslight amount remained above the beads, and this was allowed to stand atroom temperature for 1–8 hours.

To neutralize CD8+ cells in the cell sample with CD8-specific antibodies(mouse anti-human CD8), the powder of Cell Reagent (CEDARLANE) wasdissolved in 1.5 ml of PBS(−), and the total amount of this Cell Reagentsolution was mixed with 3.5–4.5 ml of the lymphocyte suspension preparedin. (15) above in a 50 ml centrifugation tube (Falcon 2070) andincubated on ice for at least 30 minutes. After adding 15 ml of PBS(−)to the lymphocyte suspension, it was centrifuging at 4° C., 200×g(approximately 1,200 rpm) for 5–10 minutes, the supernatant was removedby pipetting.

The resulting cell precipitate was washed again with 15 ml of PBS(−) andsuspended to a cell density of about 5×10⁷ cells/ml PBS (−). The beadsequilibrated with PBS(−) in a column as described above were washed with20 ml of PBS(−) adjusting the flow rate to 6–8 drops/min (1 drop/8 sec),and then the lymphocyte suspension was poured down over the columnbeads, the eluate was collected in a 15 ml tube (Falcon), and thepouring was stopped when a slight amount of the suspension remainedabove the beads. PBS (−) was further poured onto the beads, and 10–15 mlof eluate was collected. The obtained eluates were centrifuged at 4° C.at 1,200 rpm for 10 minutes, the supernatant was removed by pipetting,and the obtained precipitate was suspended to a cell density of 1.0×10⁷cells/ml in PBS(−) containing 10% fetal bovine serum (FBS).

In order to determine the ratio of CD4+ T cells in the lymphocytesuspensions, 20 μl of anti-CD4/CD8 antibody (Simultest (Leu-3A/2a),Becton Dickinson) and a control antibody (Simultest control, BectonDickinson) was mixed with 50 μl of the cell suspension in a 2 mlmicrotube and incubated on ice in the dark for 30–45 minutes, and themixture was subjected to fluorescent staining of the CD4 and CD8 on thecell surface. The mixture was diluted with 2 ml PBS(−), mixed withvortexing, centrifuged at 300×g for 5 minutes, and then the supernatantwas removed by pipetting or aspiration. The precipitate was suspended in1 ml of PBS(−), the lymphocyte suspension was applied to a flowcytometer (FACSCalibur, Becton Dickinson) and analyzed for CD4 and CD8.The results confirmed abundant CD8+ cells among the peripheral bloodlymphocytes before the column separation (FIG. 56). In contrast,although about 50% of the obtained cells from the CD4+ cell separationprocedure described above was CD4 negative or weakly positive, there wasno contamination of CD8+ cells (FIG. 59). Accordingly the purified cellswas used as CD4 positive cells for further experiments.

(17) Activation of Helper T Cells (CD4+ cells)

After adding 1 μg/ml (final concentration) of ionomycin (SIGMA) and 30nM PMA (SIGMA) to the CD4 positive cells obtained in (16) (cell density:1.0×10⁷ cells/ml), the mixture was incubated for 2 hours at 37° C. inthe presence of 5% CO₂.

(18) Fluorescent Labeling of Intracellular IL-2 mRNA of Helper T Cells(CD4+ cells)

0.9 ml of the activated CD4+ cell suspension obtained in (17) wastransferred to an electroporation cuvette (Gene Pulser specializedcuvette (electrode spacing=0.4 cm), BIO-RAD), and after adding 5.4 nmol(final concentration: 6.0 μM) of Bodipy493–503-labeled donor probe IL-2342–356(D) and 5.3 nmol (final concentration: 5.86 μM) of Cy5-labeledacceptor probe IL-2 357–371(A), a pulse was applied to the cells at 250V, 975 μF. The cell suspension was filtered through 70 μm Cell Strainer(Falcon), and after moderate centrifugation the cells were resuspendedwith PBS(−). The cell suspension was then filtered through 40 μm CellStrainer (Falcon) to remove as much of the dead cell-containing debrisas possible, centrifuged and the precipitates was resuspended withPBS(−). Some of the cells was transferred to a cover glass chamber(Lab-Tek II Chambered Coverglass #155409, NUNC Co.) and observed under afluorescence microscope to examine the ratio of fluorescing cells amongall the cells in the visual field.

-   [1] A/A {fluorescence of A (acceptor dye) emitted from cells when    the excitation light for A was irradiated to the cells}-   [2] D/A {fluorescence of A emitted from cells when the excitation    light for D (donor dye) was irradiated to the cells; FRET    fluorescence}-   [3] D/D {fluorescence of D emitted from cells when the excitation    light for D was irradiated to the cells}

Three cells in the entire visual field (25 cells) were observed emittingD/A fluorescence, indicating specific fluorescent labeling of IL-2 mRNAbased on hybridization between the mRNA and the donor and acceptorprobes. This result suggested that TH1 cells present at about 12% inactivated CD4+ cells (FIG. 60).

(19) Selective Separation of TH1 by Flow Cytometry from Activated CD4+Cells

The difference in fluorescence intensity (between IL-2 mRNA carrying andnon-carrying cells) produced by FRET fluorescence caused byhybridization of IL-2 mRNA with the donor and acceptor probe in livecells was utilized in the following attempt to selectively separate TH1.

A suspension of the fluorescent IL-2 probes-introduced cells prepared in(18) was applied to a flow cytometer (FACSCalibur). At a position inflow path, the excitation light of a donor fluorescent dye (Bodipy) wasirradiated to the cells to detect FRET fluorescence emitted fromacceptors (Cy5) caused by the hybridization, and then relativefluorescence intensity of Cy5 or Bodipy was shown as FL3-Height orFL1-Height, respectively in a dot-plots diagram. Among these plots, agroup of cells with the highest value of FL3-Height was designated as R2(FIG. 63). On the other hand, a group of typical human lymphocytes inthe points of cell size (FSC-Height; forward-scattering light) as wellas the complexity in the intrastructure (SSC-Height; side-scatteringlight) was designated as R1 according to the reference value(FACSCalibur Training Manual, BECTON DICKINSON). Cells belonging to bothR1 and R2 were selectively separated using a cell sorting function. Theseparated cells were again applied to the FACSCalibur to confirm thatthey were the objective cells. The majority of the sorted out cells bythe cell sorting function belonged to both R1 and R2, indicating thatthe cells with fluorescently labeled IL-2 mRNA had been separated out asones emitting considerable FRET fluorescence (FIG. 65). In contrast,CD4+ cells introduced no fluorescent probes were detected as dots nearat the base line of FL3-Height with only weak fluorescence at FL1-Height(FIG. 67). These results indicated that cells with specificallyfluorescent labeled IL-2 mRNA can be clearly distinguished on dot plots.

Some of the sorted out cells were then observed under a fluorescencemicroscope in the same manner as (18). All 8 cells in the entire visualfield emitted D/A (FRET fluorescence), suggesting that IL-2 mRNA wasfluorescently labeled in all the sorted out cells (FIG. 61). Comparingthe result above with that before cytometry in (18) for the ratios ofCD4+ cells with fluorescent labeled IL-2 mRNA, it was suggested that TH1had been selectively separated by the cell sorting function.

(20) Fluorescent Labeling of Intracellular IL-4 mRNA of Helper T Cells(CD4+ Cells)

0.9 ml of the activated CD4+ cell suspension obtained in (17) wastransferred to an electroporation cuvette (BIO-RAD) in the same manneras (18), and after adding 15.1 nmol (final concentration: 16.8 μM) ofBodipy493–503-labeled donor probe IL-4 265–279 and 13.6 nmol (finalconcentration: 15.1 μM) of Cy5-labeled acceptor probe IL-4 280–294, apulse was applied to the cells at 250 V, 975 μF. In the same manner as(18), the cell suspension was filterated through 70 μm Cell Strainer(Falcon), and after moderate centrifugation the cells were resuspendedwith PBS(−). To remove as much of the dead cell-containing debris aspossible, the suspension was then filtered through 40 μm Cell Strainer(Falcon), centrifuged and resuspended and then the cells weretransferred to a cover glass chamber (NUNC) and observed under afluorescence microscope to examine the ratio of A/A, D/A and D/Dfluorescing cells among the total cells in the visual field, in the samemanner as (18). Two cells in the entire visual field (41 cells) wereemitting D/A fluorescence, indicating specific fluorescent labeling ofIL-4 mRNA based on hybridization between the mRNA and the donor andacceptor probes (FIG. 68). This result suggested that TH2 is present atabout 5% in activated CD4+ cells.

(21) Selective Separation of TH2 by Flow Cytometry from Activated CD4+Cells

The difference in fluorescence intensity (between IL-4 mRNA carrying andnon-carrying cells) caused by FRET fluorescence based on hybridizationof IL-4 mRNA with the donor and acceptor probe in live cells wasutilized to selectively separate TH2.

A suspension of the fluorescent probes-introduced cells obtained in (20)was applied to a flow cytometer (FACSCalibur). The excitation light of adonor dye (Bodipy) was irradiated to the cells to detect FRETfluorescence emitted from acceptors (Cy5) based on the hybridization inthe same manner as (19), and then relative fluorescence intensity ofBodipy or Cy5 was shown as FL1-Height or FL3-Height, respectively indot-plots diagram. Among these plots, a group of cells with the highestvalue of FL3-Height was designated as R2 (FIG. 71). On the other hand, agroup of typical human lymphocytes in the points of cell size(FSC-Height; forward-scattering light) as well as the complexity in theintrastructure (SSC-Height; side-scattering light) was designated as R1(FIG. 70). Cells belonging to both R1 and R1 was selectively separatedusing a cell sorting function. The separated cells were again applied toFACSCalibur and examined to confirm that they were the objectivefluorescently labeled cells. The majority of the sorted out cells by thecell sorting function belonged to both R1 and R2 (FIGS. 72 and 73),indicating that the cells with fluorescent labeled IL-4 mRNA had beenseparated out as ones emitting considerable FRET fluorescence.

Some of the sorted-out cells were then observed under a fluorescencemicroscope in the same manner as (20). All 7 cells in the entire visualfield emitted D/A (FRET fluorescence), suggesting that IL-4 mRNA wasfluorescently labeled in all the sorted-out cells (FIG. 69). Comparingthe result above with that before cytometry in (20) for the ratios ofCD4+ cells with fluorescent labeled IL-4 mRNA, it was suggested that TH2had been selectively separated by the cell sorting function.

(22) Induction of TH2 Among Helper T Cells (CD4+ Cells)

A balance between TH1 and TH2 is maintained in healthy bodies, and it isbelieved that this balanced relationship supports homeostasis in immunesystem of the body. Conversely, disruption of the balance between TH1and TH2 is a cause of onset of numerous immune diseases. While there isno doubt that factors causing disruption in this balance in the body arethe sources responsible for such diseases, the mechanisms leading totheir onset are too complicated to reconstitute in vitro. Nevertheless,several approaches to mimic the disruption have long been performed asfollows. When TH2 is dominant over TH1, it promotes excess productionand secretion of immunoglobulins (antibody molecules) by B cells astheir humoral immune function. In this unbalanced state, the productionof autoantibodies (react with self components to cause tissue damage) isinduced to result in autoimmune diseases. An approach to mimic thiscondition by artificially preparing a TH2-dominant helper T cell groupis one utilizing the property of TH2, “IL-4 autocrine”, i.e., TH2activates itself by a cytokine (IL-4) which it produces”, whereby ahelper T cell group is treated with a high concentration of IL-4 toinduce differentiation to TH2 (Openshaw, P. et al., J. Exp. Med. 182(5),1357, 1995). Here, 20 ng/ml (final concentration) of human recombinantIL-4 (Genzyme) was added with 1 μg/ml ionomycin and 30 nM PMA asdescribed in (17) to CD4+ cells obtained in (16), and the cells wereincubated at 37° C. for 2 hours.

(23) Fluorescent Labeling of TH1 Intracellular IL-2 mRNA AmongTH2-Dominant Helper T Cells

TH1 cells were selectively separated and obtained from TH2-dominanthelper T cell group in which TH1 is sparsely present as follows. 0.9 mlof the TH2-induced CD4+ cell suspension obtained in (22) was transferredto an electroporation cuvette (BIO-RAD) in the same manner as (18).After adding 5.4 nmol (final concentration: 6.0 μM) ofBodipy493–503-labeled donor probe IL-2 342–356 and 5.3 nmol (finalconcentration: 5.86 μM) of Cy5-labeled acceptor probe IL-2 357–371, apulse was applied to the cells at 250 V, 975 μF. In the same manner as(18), the debris containing most of the dead cells was removed from thecell suspension and then some of the cells were transferred to a coverglass chamber (NUNC) and observed under a fluorescence microscope toexamine the ratio of A/A, D/A and D/D fluorescing cells among the cellsin the entire visual field. One cell in the entire visual field (23cells) was emitting D/A fluorescence, indicating the presence of a cellwith specific fluorescent labeling of IL-2 mRNA (FIG. 74). Comparingthis result with that in (18), it was suggested that TH1 is reduced toapproximately 4% in the TH2-dominant CD4+ cells from 12% in theunselectively activated CD4+ cells (FIG. 60).

(24) Selective Separation of TH1 by Flow Cytometry from TH2-DominantHelper T Cell Group

The difference in fluorescence intensity (between IL-2 mRNA carrying andnon-carrying cells) caused by FRET fluorescence based on hybridizationof IL-2 mRNA, with the donor and acceptor probe in live cells wasutilized to selectively separate TH1.

A suspension of the fluorescent probes-introduced cells obtained in (23)was applied to a flow cytometer (FACSCalibur). The excitation light of adonor dye (Bodipy) was irradiated to the cells to detect FRETfluorescence emitted from acceptors (Cy5) based on the hybridization inthe same manner as (19), and then relative fluorescence intensity ofBodipy or Cy5 was shown as FL1-Height or FL3-Height, respectively indot-plots diagram. Among these plots, a group of cells with the highestvalue of FL3-Height was designated as R2 (FIG. 77). On the other hand, agroup of typical human lymphocytes in the points of cell size(FSC-Height; forward-scattering light) as well as the complexity in theintrastructure (SSC-Height; side-scattering light) was designated as R1(FIG. 76). Cells belonging to both R1 and R2 were selectively separatedusing a cell sorting function.

The separated cells were again applied to the FACSCalibur and examinedto confirm that they were the objective fluorescently labeled cells. Themajority of the sorted out cells by the cell sorting function belongedto both R1 and R2 (FIGS. 78 and 79), indicating that the cells withfluorescent labeled IL-2 mRNA had been separated out as ones emittingconsiderable FRET fluorescence.

Some of the sorted out cells were then observed under a fluorescencemicroscope in the same manner as (18). All 5 cells in the entire visualfield emitted D/A (FRET fluorescence), suggesting that IL-2 mRNA wasfluorescently labeled in all the sorted-out cells (FIG. 75). Comparingthe result above with that before cytometry in (23) for the ratios ofCD4+ cells with fluorescent labeled IL-2 mRNA, it was suggested that TH1had been selectively separated by the cell sorting function.

(25) Induction of TH1 Among Helper T Cells (CD4+ Cells)

In contrast to (22), it is known that when TH1 is dominant over TH2, itprovokes a chronic disease such as tuberculoid leprosy (Mitra, D. K. etal. Int. Immunol. 11(11), 1801, 1999). An approach to mimic thiscondition by artificially preparing a TH1-dominant helper T cell groupis one whereby a helper T cell group is treated with the TH1-activatingcytokine IL-12 and with specific antibodies for IL-4 to neutralize andinactivate IL-4 in the extracellular fluid to induce TH1 (Openshaw, P.et al., J. Exp. Med. 182(5), 1357, 1995).

Here, 10 ng/ml (final concentration) of human recombinant IL-12(Genzyme) and 10 μg/ml anti-human IL-4 mouse monoclonal antibody(Genzyme) were added with 1 μg/ml ionomycin and 30 nM PMA as describedin (17) to CD4+ cells obtained in (16), and the cells were incubated at37° C. for 2 hours.

(26) Fluorescent Labeling of TH2 Intracellular IL-4 mRNA AmongTH1-Dominant Helper T Cells

TH2 cells were selectively separated and obtained from TH1-dominanthelper T cell group in which TH2 is sparsely present as follows. 0.9 mlof the TH1-induced CD4+ cell suspension obtained in (25) was transferredto an electroporation cuvette (BIO-RAD) in the same manner as (18).After adding 15.1 nmol (final concentration: 16.8 μM) ofBodipy493–503-labeled donor probe IL-4 265–279(D) and 13.6 nmol (finalconcentration: 15.1 μM) of Cy5-labeled acceptor probe IL-4 280–294(A), apulse was applied to the cells at 250 V, 975 μF. In the same manner as(18), the debris containing most of the dead cells was removed from thecell suspension and then some of the cells were transferred to a coverglass chamber (NUNC) and observed under a fluorescence microscope toexamine the ratio of A/A, D/A and D/D fluorescing cells among the cellsin the entire visual field. One cell in the entire visual field (42cells) was emitting D/A fluorescence, indicating the presence of a cellwith specific fluorescent labeling of IL-2 mRNA (FIG. 80). Comparingthis result with that in (18), it was suggested that TH2 is reduced toapproximately 2% in the TH1-dominant CD4+ cells from 5% in theunselectively activated CD4+ cells (FIG. 68).

(27) Selective Separation of TH2 by Flow Cytometry from TH1-DominantHelper T Cell Group

The difference in fluorescence intensity (between IL-4 mRNA carrying andnon-carrying cells) caused by FRET fluorescence based on hybridizationof IL-4 mRNA with the donor and acceptor probe in live cells wasutilized to selectively separate TH2.

A suspension of the fluorescent probes-introduced cells obtained in (26)was applied to a flow cytometer (FACSCalibur). The excitation light of adonor dye (Bodipy) was irradiated to the cells to detect FRETfluorescence emitted from acceptors (Cy5) based on the hybridization inthe same manner as (19), and then relative fluorescence intensity ofBodipy or Cy5 was shown as FL1-Height or FL3-Height, respectively indot-plots diagram. Among these plots, a group of cells with the highestvalue of FL3-Height was designated as R2 (FIG. 83). On the other hand, agroup of typical human lymphocytes in the points of cell size(FSC-Height; forward-scattering light) as well as the complexity in theintrastructure (SSC-Height; side-scattering light) was designated as R1(FIG. 82). Cells belonging to both R1 and R1 was selectively separatedusing a cell sorting function.

The separated cells were again applied to FACSCalibur and examined toconfirm that they were the objective fluorescently labeled cells. Themajority of the sorted-out cells by the cell sorting function belongedto both R1 and R2 (FIGS. 84 and 85), indicating that the cells withfluorescent labeled IL-4 mRNA had been separated out as ones emittingconsiderable FRET fluorescence.

Some of the sorted-out cells were then observed under a fluorescencemicroscope in the same manner as (20). All the cells in the entirevisual field emitted D/A (FRET fluorescence), suggesting that IL-4 mRNAwas fluorescently labeled in all the sorted-out cells (FIG. 81).Comparing the result above with that before cytometry in (26) for theratios of CD4+ cells with fluorescent labeled IL-4 mRNA, it wassuggested that TH2 had been selectively separated by the cell sortingfunction.

(28) Detection of IL-2 mRNA or IL-4 mRNA Carrying Cells Before FlowCytometry by In Situ Hybridization

Some of the cells before flow cytometry (cell sorter separation) weretransferred to a cover glass chamber (Lab-Tek II Chambered Coverglass#155409, NUNC Co.), and after fixing the cells with 4%paraformaldehyde/PBS (pH 7.4) at room temperature for 30 minutes, theFISH (Fluorescence in situ Hybridization) method described below wasused to determine the ratio of cells carrying IL-2 mRNA or IL-4 mRNA(IL-2 mRNA(+) or IL-4 mRNA(+)) present among the cells in the entirevisual field.

First, a digoxigenin (hereunder, DIG)-labeled RNA probe for IL-2 RNA todetect intracellular IL-2 mRNA of the fixed cells was synthesized usinga DIG RNA Labeling Kit (Boehringer Mannheim) according to the protocolof the kit manual. 10 μg of recombinant plasmid (pTCGF#2) constructedfor human IL-2 RNA synthesis by the method described in (2A) wascompletely linearized by EcoRI digestion, and the linearized DNA in theobtained DNA solution was extracted with phenol/chloroform fordenaturation and removal of protein and then purified by ethanolprecipitation, to prepare a template for RNA probe synthesis. Thistemplate DNA (5 μg) was mixed with 1.8 mM ATP, 0.9 mM CTP, 0.7 mM GTP,1.1 mM UTP and 0.58 mM UTP (DIG-labeled) in the presence of T7 RNApolymerase and incubated at 37° C. for 2 hours. After DNaseI solutionwas added to the solution and incubated for 10 minutes to degrade thetemplate DNA, a 1/10 volume of 5 M sodium acetate and an equal volume ofisopropanol were added to this reaction solution and centrifuged at15,000 g×15 min to recover the synthesized RNA a precipitate. Theprecipitate was dissolved in RNase-free sterile distilled water.

Secondly, a DIG-labeled RNA probe for IL-4 RNA to detect intracellularIL-4 mRNA of the fixed cells was synthesized using a DIG RNA LabelingKit (Boehringer Mannheim) in the same manner as the IL-2. A recombinantplasmid (phIL-4#9) constructed for human IL-4 RNA synthesis by themethod described in (2B) was linearized by complete digestion with SmaI,and the linearized DNA was treated with phenol/chloroform and thenpurified by ethanol precipitation, to prepare a template for RNA probesynthesis. This template DNA (5 μg) was mixed with 1.2 mM ATP, 1.0 mMCTP, 1.1 mM GTP, 0.8 mM UTP and 0.5 mM UTP (DIG-labeled) in the presenceof T7 RNA polymerase and incubated at 37° C. for 2 hours. After DNaseIsolution was added and reacted for 10 minutes to degrade the templateDNA, a 1/10 volume of 5 M sodium acetate and an equal volume ofisopropanol were added to this reaction solution, and centrifuged at15,000 g×15 min to recover the synthesized RNA as a precipitate. Theprecipitate was dissolved in RNase-free sterile distilled water.

Thirdly, highly fragmented RNA probes for IL-2 or IL-4 were prepared touse for a hybridization experiment as in (13) because the introductionof the full length IL-2 or IL-4 RNA probe obtained above was thought tocause high background noise due to the remaining non-hybridized RNAprobe in the cells. 10 μg of the IL-2 or IL-4 RNA probe was dissolved in100 μl of an alkaline solution (42 mM NaHCO₃, 63 mM Na₂CO₃, 5 mM DTT)and incubated at 60° C. for 10–15 minutes. 10 μl of 3 M sodium acetateand 350 μl of EtOH were added to precipitate the RNA. After standing at−20° C. for 30 minutes, it was centrifuged at 16 krpm for 20 minutes.The precipitate was washed with 70% ethanol, dried, and dissolved in 50μl of RNase-free sterile distilled water to prepare an alkalinedenatured IL-2 or IL-4 RNA probe.

The cells fixed on the bottom of the chamber were washed 3 times withPBS(−), treated with a 0.1% Triton X-100/PBS solution at roomtemperature for 5 minutes to permeabilize the cells, and then washed 3times with PBS(−) and treated with 0.2 N HCl at room temperature for 10minutes. After washing the cell monolayer with PBS(−), it was incubatedfor 5 minutes at 37° C. with 1 μg/ml of Proteinase K/PBS solution. Afterwashing the cell monolayer with PBS(−), it was fixed again with 4%paraformaldehyde/PBS (pH 7.4) for 30 minutes. This was washed twice with2 mg/ml of glycine/PBS (15 minutes per washing) and treated with 50%deionized formaldehyde/2×SSC solution (hereunder, Soln. A) for 30minutes to prepare a hybridization solution (50% deionized formaldehyde,5× Denhardt, 2×SSC, alkaline denatured IL-2 or IL-4 RNA probe (1μg/ml)).The solution was denatured at 90° C. for 10 minutes and cooled on ice.Adding 100 μl of the solution to the cells, they were incubated forhybridization overnight at 45° C.

The cell monolayer after hybridization was washed with Soln. A for 5minutes at 45° C., and then washed twice with Soln. B (10 mM Tris.HCl(pH 8.5), 500 mM NaCl) (5 min/washing) and treated with 20 mg/ml RNaseA/Soln. B (pretreated at 90° C. for 10 minutes) at 37° C. for 20minutes. After washing with Soln. A, and then Soln. C (50% deionizedformaldehyde/1×SSC) for 30 minutes each at 45° C., the cells were washedagain with Soln. C at room temperature for 20 minutes. After washingwith Buffer 1 (100 mM maleic acid, 150 mM NaCl (pH 7.5)) (2×5 min), itwas subjected to blocking with Buffer 2 (1% Blocking Reagent (BoehringerMannheim) in Buffer 1) at room temperature for 20 minutes. After washingtwice with Buffer 1, FITC-labeled anti-DIG antibody (Fab, diluted withBuffer 2 by 100 times, protein concentration: approximately 1 μg/ml) wasadded to the cells and incubated for at least 30 minutes. After washing3 times with PBS(−), the cells were observed under a fluorescencemicroscope to examine the ratio of IL-2 mRNA or IL-4 mRNA carrying cells(TH1 or TH2) among the cells in the entire visual field.

In the cells used for the experiment of (18) in which IL-2 mRNA wasfluorescently labeled for selective separation of TH1, 5 cells werefound to carry IL-2 mRNA out of 24 cells and 3 cells out of 43 cellswere carrying IL-4 mRNA (FIG. 86). The ratio of IL-2 mRNA carrying cells(20.8%) was somewhat higher than that in (18) (12.0%). This gap isthought to be caused by the difference in the way of probe-introduction,i.e., the fluorescent probes were not introduced to some of TH1 cells inthe experiment of (18).

In the cells for the experiment of (20) in which IL-4 mRNA wasfluorescently labeled for selective separation of TH2, 6 cells werefound to carry IL-2 mRNA out of 28 cells and 3 cells out of 42 cellswere carrying IL-4 mRNA (FIG. 88). The ratio of IL-4 mRNA carrying cells(7.1%) was slightly higher than that in (20) (4.9%).

Furthermore, in the cells used for the experiments of (23) in which IL-2mRNA was fluorescently labeled for selective separation of TH1 from aTH2-dominant cell group, 2 cells out of 28 cells and 3 cells out of 35cells were found to carry IL-2 mRNA, while 8 cells out of 41 cells and 9cells out of 42 cells were carrying IL-4 mRNA (FIG. 90). The ratio ofIL-2 mRNA carrying cells (7.9%) was higher than that in (23) (4.3%).This gap is thought to be caused by the difference in the way ofprobe-introduction, i.e., the fluorescent probes were not introduced tosome of TH1 cells in the experiments of (23).

Also, in the cells used for the experiments of (26) in which IL-4 mRNAwas fluorescently labeled for selective separation of TH2 from aTH1-dominant cell group, 9 cells out of 24 cells and 11 cells out of 29cells were found to carry IL-2 mRNA, while 1 cells out of 48 cells and 1cells out of 41 cells were carrying IL-4 mRNA (FIG. 92). The ratio ofIL-4 mRNA carrying cells (2.2%) was equal to that in (26) (2.4%). Thisconsistence between the fluorescence microscope observation results andFISH results for IL-4 mRNA would be due to much the same frequency ofprobe-introduction, i.e., much the same amount of fluorescent probeswere introduced to almost all the TH2 cells. This relatively uniformedintroduction is thought to be obtained by the application of electricalpulse to the cells in the presence of a higher concentration of IL-4fluorescent probe compared with IL-2 probe.

(29) Detection of IL-2, γ-IF, TNF-β, IL-4, IL-5 and IL-10 mRNA CarryingCells in the Cells After Flow Cytometry (Selectively Separated with aCell Sorter) by in Situ Hybridization

Some of the cells after flow cytometry (selectively separated with acell sorter) obtained in (24) to (27) were transferred to a cover glasschambers (NUNC). After fixing the cells with 4% paraformaldehyde/PBS (pH7.4) at room temperature for 30 minutes, the FISH method described inthe detail in (28) was used to examine the ratios of CD4+ cells havingmRNA for the TH1 cytokines IL-2, γ-IF, TNF-β and the TH2 cytokines IL-4,IL-5 and IL-10. Since alkaline denatured RNA probes for IL-2 and IL-4had already been obtained in (28), DIG-labeled RNA probes to detect mRNAfor each of the cytokines other than IL-2 and IL-4 were synthesizedusing a DIG RNA Labeling Kit (Boehringer Mannheim) according to theprotocol of the kit manual.

First, for γ-IF, plasmid DNA (pPLc28-HIIF52) including human γ-IF cDNAwas extracted and purified using a Plasmid Midi Kit (QIAGEN) from theplasmid-carrying E. coli (ATCC#39278) that had been cultured at 28° C.The plasmid was digested with restriction enzymes BamHI and ClaI, andthe obtained γ-IF cDNA fragment was linked to the AccI and BamHIrestriction site of a pBluescript KS(+) vector for RNA synthesis usingDNA Ligation kit version 2 (Takara). The DNA solution was introducedinto competent cells of E. coli JM109 (Takara) and the recombinantplasmid DNA was extracted and purified from 100 ml culture of theresulting E. coli transformants using a Plasmid Midi Kit (QIAGEN). Therecombinant plasmid (phγ-IF#1) was digested with restriction enzymeKpnI. After the linearized plasmid DNA was treated withphenol/chloroform, it was purified by ethanol precipitation to prepare atemplate for RNA probe synthesis. This template DNA (5 μg) was mixedwith 1.3 mM ATP, 0.7 mM CTP, 0.8 mM GTP, 0.8 mM UTP and 0.43 mM UTP(DIG-labeled) in the presence of T7 RNA polymerase and incubated at 37°C. for 7 hours. After adding DNaseI to the reaction mixture, it wasincubated for 10 minutes to degrade the template DNA. After a 1/10volume of 5 M sodium acetate and an equal volume of isopropanol wereadded to the RNA solution, it was centrifuged at 15,000 g×15 min torecover the synthesized RNA as a precipitate. The precipitate wasdissolved in RNase-free sterile distilled water.

Secondly, for TNF-β, plasmid DNA carrying human TNF-β cDNA was extractedand purified using a Plasmid Midi Kit (QIAGEN) from 50 ml culture of E.coli HILBI37 (ATCC#104607) carrying the plasmid. The plasmid wasdigested with restriction enzyme BamHI. The linearized plasmid DNA wastreated with phenol/chloroform and purified by ethanol precipitation toprepare a template for RNA probe synthesis. The template DNA (5 μg) wasmixed with 0.7 mM ATP, 1.4 mM CTP, 1.1 mM GTP, 0.4 mM UTP and 0.24 mMUTP (DIG-labeled) in the presence of T7 RNA polymerase and incubated at37° C. for 6 hours. After adding DNaseI to the reaction mixture, it wasincubated for 10 minutes to degrade the template DNA. After adding a1/10 volume of 5 M sodium acetate and an equal volume of isopropanol tothe RNA solution, it was centrifuged at 15,000 g×15 min to recover thesynthesized RNA as a precipitate. The precipitate was dissolved inRNase-free sterile distilled water.

Thirdly, for IL-5, 2 μg of lyophilized human IL-5 cDNA-containingplasmid DNA, phIL-5–115.1 (ATCC#59395), was dissolved in 2 μl of steriledistilled water. 1 ng of the plasmid was introduced into competent cellsof E. coli JM109 (Takara) and the plasmid DNA was extracted and purifiedusing a Plasmid Midi Kit (QIAGEN) from 50 ml culture of the resulting E.coli transformants. The plasmid DNA was digested with restriction enzymeBamHI, and the isolated IL-5 cDNA fragment was linked to the BamHIrestriction site of a pBluescript KS(+) vector for RNA synthesis usingDNA Ligation kit version 2 (Takara). The DNA solution was introducedinto competent cells of E. coli JM109 (Takara). The recombinant plasmidDNA was extracted and purified from 100 ml culture of the resulting E.coli transformants using a Plasmid Midi Kit(QIAGEN). The recombinantplasmid (phIL-5#8) was digested with restriction enzyme NotI. Aftertreating the linearized DNA with phenol/chloroform, it was purified byethanol precipitation to prepare a template for RNA probe synthesis.This template DNA (5 μg) was mixed with 1.3 mM ATP, 0.7 mM CTP, 0.8 mMGTP, 0.8 mM UTP and 0.42 mM UTP (DIG-labeled) in the presence of T3 RNApolymerase and incubated at 37° C. for 6 hours. Adding DNaseI to thereaction mixture, it was incubated for 10 minutes to degrade thetemplate DNA. After adding a 1/10 volume of 5 M sodium acetate and anequal volume of isopropanol were added to this reaction solution, it wascentrifuged at 15,000 g×15 min to recover the synthesized RNA as aprecipitate. The precipitate was dissolved in RNase-free steriledistilled water.

Fourthly, for IL-10, pH15C, a plasmid DNA containing human IL-10 cDNAwas extracted and purified using Plasmid Midi Kit (QIAGEN) from 50 mlculture of an E. coli strain (ATCC#104607) carrying the plasmid. Theplasmid was digested with restriction enzyme BamHI, and the isolatedIL-10 cDNA fragment was linked to the BamHI restriction site of apBluescript KS(+) vector for RNA synthesis using a DNA Ligation kitversion 2 (Takara). The recombinant plasmid was introduced intocompetent cells of E. coli JM109 (Takara). The recombinant plasmid DNAwas extracted and purified from 50 ml culture of the resulting E. Colitransformants using a Plasmid Midi Kit (QIAGEN). The recombinant plasmid(phIL-10#10) was digested with restriction enzyme SmaI. After treatingthe linearized DNA with phenol/chloroform, it was purified by ethanolprecipitation to prepare a template for RNA probe synthesis. Thistemplate DNA (5 μg) was mixed with 1.1 mM ATP, 0.9 mM CTP, 0.9 mM GTP,0.6 mM UTP and 0.50 mM UTP (DIG-labeled) in the presence of T7 RNApolymerase and incubated at 37° C. for 6 hours. Adding DNaseI to thereaction mixture, it was incubated for 10 minutes to degrade thetemplate DNA. After adding a 1/10 volume of 5 M sodium acetate and anequal volume of isopropanol to this reaction solution, it wascentrifuged at 15,000 g×15 min to recover the synthesized RNA as aprecipitate. The precipitate was dissolved in RNase-free steriledistilled water.

The full-length RNA probes for γ-IF, TNF-β, IL-5 and IL-10 obtainedabove were highly fragmented in the same manner as those for IL-2 andIL-4. 10 μg of each RNA probe was dissolved in 10 μl of theabove-mentioned alkali-denaturing solution and incubated at 60° C. for10–15 minutes. Adding 10 μl of 3 M sodium acetate and 350 μl of ethanolto the denaturing solution, it was cooled at −20° C. for 30 minutes andcentrifuged at 16 krpm for 20 minutes to precipitate the RNA probe. Theprecipitate was rinsed with 70% ethanol, dried, and dissolved in 50 μlof RNase-free sterile distilled water to prepare an alkaline denaturedγ-IF, TNF-β, IL-5 or IL-10 RNA probe. Washing the cells fixed on thebottom of the chamber 3 times with PBS(−), the cells were treated with a0.1% Triton X-100/PBS solution at room temperature for 5 minutes topermeabilize the cells. Washing 3 times with PBS(−), the permeabilizedcells were treated with 0.2 N HCl at room temperature for 10 minutes.After washing the cells with PBS(−), it was incubated for 5 minutes at37° C. with 1 μg/ml Proteinase K in PBS(−). Washing the resulting cellswith PBS(−), it was fixed again with 4% paraformaldehyde/PBS (pH 7.4)for 30 minutes. Washing the fixed cells twice with 2 mg/ml glycine inPBS for 15 minutes each, they were treated with 50% deionizedformaldehyde/2×SSC solution (hereunder, Soln. A) for 30 minutes toprepare a hybridization solution (50% deionized formaldehyde, 5×Denhardt, 2×SSC, alkaline denatured IL-2, γ-IF, TNF-β, IL-4, IL-5, orIL-10 RNA probe (1 μg/ml)). Denaturing the solution at 90° C. for 10minutes, it was cooled on ice. Adding 100 μl of the same solution to thecells, they were incubated overnight at 45° C.

After the hybridization, the cells were washed with Soln. A for 5minutes at 45° C., and then washed twice with Soln. B (10 mM Tris.HCl(pH 8.5), 500 mM NaCl) for 5 minutes each. The cells were treated with20 mg/ml RNase A/Soln. B (pretreated at 90° C. for 10 minutes) at 37° C.for 20 minutes. After washing the cells with Soln. A and Soln. C (50%deionized formaldehyde/1×SSC) for 30 minutes each at 45° C., they werewashed with Soln. C at room temperature for 20 minutes. Washing thecells with Buffer 1 (100 mM maleic acid, 150 mM NaCl (pH 7.5)) (2×5min), they were treated with Buffer 2 (1% Blocking Reagent (BoehringerMannheim) in Buffer 1) at room temperature for 20 minutes. After washingthe cells twice with Buffer 1, FITC-labeled anti-DIG antibody (Fab,diluted with Buffer 2 by 100 times, protein concentration: approximately1 μg/ml) was added to the cells and they were incubated for at least 30minutes. Washing the cells 3 times with PBS(−), they were observed undera fluorescence microscope to examine the ratio of IL-2, γ-IF, TNF β,IL-4, IL-5 or IL-10 mRNA carrying cells (TH1 or TH2) among the cells inthe entire visual field. The cell type (TH1 or TH2) was determined forthe selectively separated cells by a cell sorting function.

The cells selectively separated by the cell sorter in (19) and (24) werepositive for IL-2, γ-IF and TNF-β mRNA and negative for IL-4, IL-5 andIL-10 (FIGS. 87 and 91), indicating that all the cells examined wereTH1-specific cytokine-producing cells. On the other hand, the cellsselectively separated by the cell sorter in (21) and (27) were negativefor IL-2, γ-IF and TNF-β mRNA and positive for IL-4, IL-5 and IL-10(FIGS. 89 and 93), identifying all the cells examined as TH2-specificcytokine-producing cells.

(30) Selective Separation of Live TH1 and TH2 Cells Expressing SpecificGenes Based on Difference in Fluorescence Intensity

All the selective separation experiments of TH1 or TH2 cells based onthe fluorescently labeled IL-2 or IL-4 mRNA were summarized in Table 9.In this table, their mRNAs were utilized as markers to isolate theobjective TH cells throughout the experimental results from (18), (20),(23) and (26).

TABLE 9 Flow Cytometry Before IL-2 or After FRET IL-4 FRET Mixing Ratios(D/A) mRNA (D/A) IL-2 or Target positive carrying positive IL-4 mRNAcell cells cells cells carrying No. Marker type (%) (%) (%) cells (%)(18) IL-2 TH-1 12.0 20.8 100 100 mRNA (20) IL-4 TH-2 4.9 7.1 100 100mRNA (23) IL-2 TH-1 4.3 7.9 100 100 mRNA (26) IL-4 TH-2 2.4 2.2 100 100mRNA

In (18), the IL-2 expressing cells (TH1) which presented only at 12%(FIG. 60, live cell fluorescent observation results) or 20% (FIG. 86,FISH experiment results) were concentrated to 100% by the separationmethod utilizing the difference in fluorescence intensities as describedin (19). The IL-4 expressing cells (TH2) presented only at 4.9% (FIG.68, fluorescent observation results) or 7.1% (FIG. 88, FISH experimentresults) before flow cytometry as described in (20). However, all thecells obtained by the selective separation method of (21) were IL-4 mRNAcarrying cells.

Furthermore, the ratio of TH1 to TH2 cells was artificially shiftedtoward TH2 to mimic immune diseases with overactivation of TH2 cells in(23). Compared with (18), TH1 was notably reduced to 4.3% (FIG. 74,fluorescent observation results) or 7.9% (FIG. 90, FISH experimentresults). However, TH1 cells were obtained at 100% purity by theselective separation method as shown in (24). In contrast to (23), thebalance between TH1 and TH2 was artificially shifted toward TH1 in (26)to mimic immune diseases with overwhelming presence of TH1. Comparedwith (25), TH2 cells were reduced to 2.4% (FIG. 80, fluorescentobservation results) or 2.3% (FIG. 92, FISH experiment results).However, TH2 cells were obtained at 100% purity by the selectiveseparation method as shown in (27).

To confirm the above-mentioned results, mRNA of TH1- or TH2-specificcytokine was detected in (29). It was demonstrated that all the cellsseparated by the cell sorter in (19) and (24) from the cells of (18) and(23) were TH1, while all the cells selectively separated in (21) and(27) from the cells of (20) and (26) were TH2.

Throughout all these results, it was concluded that TH1 or TH2 cells areselectively separated with complete selectivity (100%) from cellscontaining the both cell types by the separation method utilizing mRNAof IL-2 or IL-4, a specific cytokine for TH1 or TH2 cells, respectively.

From the invention thus described, it will be obvious that the inventionmay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedfor inclusion within the scope of the following claims.

1. A method for selectively separating live cells which have expressed aspecific mRNA from a live cell group comprising: a first step ofdetermining a site of the specific mRNA that has high accessibility foroligonucleotide probe hybridization and preparing an oligonucleotideprobe or probe set, labeled with a fluorescent dye or fluorescent dyes,having a base sequence or base sequences capable of hybridizing to thebase sequence of the thus determined site, wherein the fluorescence ofthe fluorescent dye or fluorescent dyes will change upon the formationof a hybrid between the labeled oligonucleotide probe or probe set andthe specific mRNA; a second step of introducing the labeledoligonucleotide probe or probe set into cells in a live cell groupcontaining the live cells which have expressed the specific mRNA and thelive cells which have not expressed the specific mRNA, whereby thelabeled oligonucleotide probe or probe set hybridizes to the specificmRNA expressed in the live cells; a third step of irradiating light tothe live cell group containing the live cells having hybridized andunhybridized oligonucleotide probe or probe set and the live cellshaving only unhybridized oligonucleotide probe or probe set andmeasuring the fluorescence which is emitted by the live cells, whereinthe fluorescence from the cells having hybridized and unhybridizedoligonucleotide probe or probe set is different from the fluorescencefrom the cells having only unhybridized oligonucleotide probe or probeset due to a change in fluorescence caused by hybrid formation, toidentify the live cells wherein the hybrid formation of the labeledoligonucleotide probe or probe set and the specific mRNA has takenplace; and a fourth step of separating the identified live cells fromthe live cell group.
 2. The method according to claim 1, wherein theprobe set comprises a first probe and a second probe, the first probeand the second probe have base sequences capable of hybridizing to saidmRNA adjacent to each other, and the first probe is labeled with anenergy donor fluorescent dye and the second probe is labeled with anenergy acceptor fluorescent dye, and the change in fluorescence iscaused by fluorescence resonance energy transfer (FRET) from the energydonor fluorescent dye of the first probe to the energy acceptorfluorescent dye of the second probe.
 3. The method according to claim 1,wherein the selective separation in the fourth step of the identifiedlive cells based on the change in fluorescence is performed by a cellsorter.
 4. The method according to claim 1, wherein the specific mRNA isa mRNA encoding a cytokine.
 5. The method according to claim 1, whereinthe live cells selectively separated in the fourth step are T Helper 1(TH1) cells.
 6. The method according to claim 1, wherein the live cellsselectively separated in the fourth step are T Helper 2 (TH2) cells. 7.A method for selectively separating live cells which have expressed amRNA encoding human interleukin-2 (IL-2) comprising: a first step ofintroducing a probe or probe set into cells in a live cell groupcontaining live cells which have expressed the mRNA; wherein the probeor probe set has a base sequence or base sequences capable ofspecifically hybridizing to a human IL-2 mRNA and is labeled with afluorescent dye or fluorescent dyes, wherein the fluorescence of thefluorescent dye or fluorescent dyes will change upon the formation of ahybrid between the labeled oligonucleotide probe or probe set and themRNA; whereby the labeled oligonucleotide probe or probe set hybridizesto the specific mRNA expressed in the live cells; a second step ofirradiating light to the live cell group containing the live cellshaving hybridized and unhybridized oligonucleotide probe or probe setand the live cells having only unhybridized oligonucleotide probe orprobe set and measuring the fluorescence which is emitted by the livecells, wherein the fluorescence from the cells having hybridized andunhybridized oligonucleotide probe or probe set is different from thefluorescence from the cells having only unhybridized oligonucleotideprobe or probe set due to a change in fluorescence caused by hybridformation, to identify the live cells wherein the hybrid formation ofthe labeled oligonucleotide probe or probe set and the mRNA has takenplace; and a third step of selectively separating the identified livecells from the live cell group.
 8. The method according to claim 7,wherein the probe set comprises a first probe and a second probe, thefirst probe and the second probe have base sequences capable ofhybridizing to said mRNA adjacent to each other, and the first probe islabeled with an energy donor fluorescent dye and the second probe islabeled with an energy acceptor fluorescent dye, and said change influorescence is caused by fluorescence resonance energy transfer (FRET)from the energy donor fluorescent dye of the first probe to the energyacceptor fluorescent dye of the second probe.
 9. A method forselectively separating live cells which have expressed a mRNA encodinghuman interleukin-4 (IL-4) comprising: a first step of introducing aprobe or probe set into cells in a live cell group containing live cellswhich have expressed the mRNA; wherein the probe or probe set has a basesequence or base sequences capable of specifically hybridizing to ahuman IL-4 mRNA and is labeled with a fluorescent dye or fluorescentdyes, wherein the fluorescence of the fluorescent dye or fluorescentdyes will change upon the formation of a hybrid between the labeledoligonucleotide probe or probe set and the mRNA; whereby the labeledoligonucleotide probe or probe set hybridizes to the specific mRNAexpressed in the live cells; a second step of irradiating light to thelive cell group containing the live cells having hybridized andunhybridized oligonucleotide probes and the live cells having onlyunhybridized oligonucleotide probes and measuring the fluorescence whichis emitted by the live cells, wherein the fluorescence from the cellshaving hybridized and unhybridized oligonucleotide probes is differentfrom the fluorescence from the cells having only unhybridizedoligonucleotide probes due to a change in fluorescence caused by hybridformation, to identify the live cells wherein the hybrid formation ofthe labeled oligonucleotide probes and the mRNA has taken place; and athird step of selectively separating the identified live cells from thelive cell group.
 10. The method according to claim 9, wherein the probeset comprises a first probe and a second probe, the first probe and thesecond probe have base sequences capable of hybridizing to said mRNAadjacent to each other, and the first probe is labeled with an energydonor fluorescent dye and the second probe is labeled with an energyacceptor fluorescent dye, and said change in fluorescence is caused byfluorescence resonance energy transfer (FRET) from the energy donorfluorescent dye of the first probe to the energy acceptor fluorescentdye of the second probe.
 11. The method according to claim 7, whereinthe probe or probe set is capable of hybridizing to the 287–316 site orthe 342–371 site of human IL-2 mRNA.
 12. The method according to claim8, wherein the probe set is capable of hybridizing to the 287–316 siteor the 342–371 site of human IL-2 mRNA and includes a probe setcorresponding to the SEQ ID NO: 7 and 8 or SEQ ID NO: 9 and
 10. 13. Themethod according to claim 9, wherein the probe or probe set is capableof hybridizing to the 176–205 site or the 265–294 site of human IL-4mRNA.
 14. The method according to claim 10, wherein the probe set iscapable of hybridizing to the 176–205 site or the 265–294 site of humanIL-4 mRNA and includes a probe set corresponding to the SEQ ID NO: 15and 16 or SEQ ID NO: 17 and 18.